Compositions for Treating Ectopic Calcification Disorders, and Methods Using Same

ABSTRACT

The present invention includes compositions and methods for treating disease and disorders associated with pathological calcification or pathological ossification.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/257,883, filed Nov. 20, 2015, whichapplication is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Calcification is the accumulation of calcium salts in a body tissue. Itnormally occurs during formation of bone, but calcium can also bedeposited abnormally in soft tissues such as arteries, cartilage andheart valves. Vascular calcification frequently develops in patientswith atherosclerosis, stroke, valvular disease and varicosis. Advancedage and metabolic disorders, including diabetes mellitus arecontributing factors.

Ossification refers to the process of bone tissue formation or boneremodeling orchestrated by the osteoblasts. Ossification allows bones toform while a fetus is still in the womb, and also converts various typesof connective tissue into bone. The two main processes of ossificationare intra-membranous ossification and intra-cartilaginous ossification,which differ based on the area of the body in which the cartilage islocated.

Abnormalities in the levels of calcification and ossification lead to aspectrum of diseases, a few examples of such as general arterialcalcification of infancy (GACI), idiopathic infantile arterialcalcification (IIAC), pseudoxanthoma elasticum (PXE), ossification ofposterior longitudinal ligament (OPLL), medial wall vascularcalcification (MWVC), autosomal recessive hypophosphatemia ricketstype-2 (ARHR2), end state renal disease (ESRD), chronic kidneydisease-bone/mineral disorder (CKD-MBD), X-linked hypophosphatemia(XLH), age related osteopenia, calcific uremic arteriolopathy (CUA) andhypophosphatemic rickets.

GACI is an ultra-rare neonatal disease characterized by infantile onsetof widespread arterial calcifications in large and medium sized vessels,resulting in cardiovascular collapse and death in the neonatal period.The disease presents clinically with heart failure, respiratorydistress, hypertension, cyanosis, and cardiomegaly. The prognosis isgrave, with older reports of a mortality rate of 85% at six months,while recently intensive treatment with bisphosphonates (such asetridonate) has lowered mortality to 55% at six months. Tempering thisapparent progress is the severe skeletal toxicity associated withprolonged use of etridonate in patients with GACI, and theineffectiveness of bisphosphonates to prevent mortality in some patientseven when instituted early. Further, the limited available data makes itdifficult to determine if bisphosphonate treatment is truly protectiveor reflects the natural history of the disease in less effectedpatients. Interestingly, serum PPi levels appear to be significantlydepleted in GACI patients.

Kidneys are integral to maintenance of normal bone and mineralmetabolism, including excretion of phosphate. In 2003, 19.5 million U.S.adults have chronic kidney disease (CKD), and 13.6 million had stage 2-5CKD, as defined by the National Kidney Foundation Kidney DiseaseOutcomes Quality Initiative (NKFK/DOQI). The prevalence of ESRD isincreasing at an alarming rate. In 2000, end stage kidney diseasedeveloped in over 90,000 people in the U.S. The population of patientson dialysis therapy or needing transplantation was 380,000 in 2003, andbecame 651,000 patients in 2010. Care for patients with ESRD alreadyconsumes more than $18 billion per year in the U.S., a substantialburden for the health care system. Importantly, patients with kidneyfailure are unable to appropriately regulate serum mineral balance andtend to retain phosphate that is absorbed from the various dietarycomponents. A high serum level of phosphate is associated with excessivesecretion of parathyroid hormone and a tendency to calcification of thesoft tissues, including blood vessels.

In patients with kidney failure, excess removal of phosphate andpyrophosphate anions can occur during hemodialysis or peritonealdialysis. Depletion of these anions from tissues and plasma leads todisorders of bone and mineral metabolism, including osteomalacia andcalcification of soft tissues and bone disease. Deposition of calciuminto the small vessels of the skin causes an inflammatory vasculitiscalled calciphylaxis, which can lead to gangrene of the skin andunderlying tissues, resulting in severe, chronic pain. Calciphylaxis maynecessitate amputation of the affected limb and is commonly fatal, withno effective treatment for this condition. It is thus important toregulate the amount of pyrophosphate in the system and reduce theoccurrence of calciphylaxis in patients.

CUA is a fatal disease seen in patients with CKD on dialysis.Calcification of small arteries leads to tissue/skin ischemia,infarction and thrombosis, with patient mortality close to 80%.Currently there are 450,000 patients on dialysis in the U.S. who are atrisk of acquiring CUA, and there is no FDA approved treatments for thedisease. CUA has hallmarks resembling GACI and other disorders ofcalcification, exhibiting low levels of PPi and high levels offibroblast growth factor 23 (FGF23). In ESRD patients requiringdialysis, this calcification process is further accelerated, with anaverage life-expectancy of 5-6 years.

PXE is a heritable disorder characterized by mineralization of elasticfibers in skin, arteries and the retina, which results in dermal lesionswith associated laxity and loss of elasticity, arterial insufficiency,cardiovascular disease and retinal hemorrhages leading to maculardegeneration. Mutations associated with PXE are also located in theabcc6 gene. Characteristic skin lesions (yellowish papules and plaquesand laxity with loss of elasticity, typically seen on the face, neck,axilla, antecubital fossa, popliteal fossa, groin and periumbilicalareas) are generally an early sign of PXE and result from anaccumulation of abnormal mineralized elastic fibers in the mid-dermis.They are usually detected during childhood or adolescence and progressslowly and often unpredictably. A PXE diagnosis can be confirmed by askin biopsy that shows calcification of fragmented elastic fibers in themid- and lower dermis. The skin manifestations are among the most commoncharacteristics of PXE, but the ocular and cardiovascular symptoms areresponsible for the morbidity of the disease.

Common cardiovascular complications of PXE are due to the presence ofabnormal calcified elastic fibers in the internal elastic lamina ofmedium-sized arteries. The broad spectrum of phenotypes includespremature atherosclerotic changes, intimal fibroplasia causing angina orintermittent claudication or both, early myocardial infarction andhypertension. Fibrous thickening of the endocardium and atrioventricularvalves can also result in restrictive cardiomyopathy. Approximately 10%,of PXE patients also develop gastrointestinal bleeding and centralnervous system complications (such as stroke and dementia) as aconsequence of systemic arterial wall mineralization. In addition,renovascular hypertension and atrial septal aneurysm can be seen in PXEpatients.

Conditions in which serum phosphate levels are reduced or elevated arereferred to as hypophosphatemia and hyperphosphatemia, respectively.Hypophosphatemia, which often results from renal phosphate wasting, iscaused by a number of genetic disorders including X-linkedhypophosphatemic rickets (XLH), hereditary hypophosphatemic rickets withhypercakiuria (HHRH), hypophosphatemic bone disease (HBD), and autosomaldominant hypophosphatemic rickets (ADHR). The exact molecular mechanismsby which proper serum phosphate concentrations are maintained are poorlyunderstood.

There is a need in the art for novel compositions and methods fortreating diseases and disorders associated with pathologicalcalcification and/or pathological ossification. Such compositions andmethods should not undesirably disturb other physiologic processes. Suchcompositions and methods should reduce the level of calcification andincreasing PPi plasma levels in individuals who exhibit lower thannormal plasma PPi levels. The present invention fulfills this need.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of exemplary embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings exemplary embodiments. It should beunderstood, however, that the invention is not limited to the precisearrangements and instrumentalities of the embodiments shown in thedrawings.

FIGS. 1A-1C comprise graphs illustrating studies of human ENPP3 steadystate ATP hydrolysis activity. FIG. 1A illustrates time courses of AMPproduct formation after addition of 50 nM hNPP3 with (from bottom totop) 0.98, 1.95, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 μM ATP.The enzyme reaction was quenched by equal volume of 3 M formic acid atdifferent times, and the reaction product AMP was quantified by HPLCanalysis with an AMP standard curve. The smooth line though data pointsare best fits to a non-linear enzyme kinetic model with productinhibition and substrate depletion. FIG. 1B illustrates steady stateATPase cycling rate comparison. ENPP3 substrate concentration dependenceof initial steady state enzyme cycling rate was compared with thepreviously measured values for human ENPP1. ATPase cycling reaction ofboth 50 nM hNPP3 and hNPP1 totally depleted ATP substrate in 1 minutefor 0.98, 1.95 and 3.9 μM ATP, and thus these three rates were omittedfrom the plot because their rates could not be accurately determined.The hNPP3 steady state ATPase reaction reached the maximum (k_(cat)) of2.59(±0.04) s⁻¹ enzyme⁻¹, from the weighted average of the measuredrates with 7.8, 15.6, 31.3, 62.5, 125 μM substrate concentration,seeming slower than that for hNPP1 3.46(±0.44) s⁻¹ enzyme⁻¹. The K_(M)can be estimated <8 μM. At substrate [ATP]>125 μM, hNPP3 ATPase cyclingrate gradually decreased. FIG. 1C illustrates substrate concentrationdependent η. The decreasing η value with substrate concentration forboth enzymes indicates that substrate depletion contributes to thenon-linearity in the enzyme reaction time courses much more than productinhibition at the lower initial substrate concentration. The strikingsimilarity with human ENPP3 vs. human ENPP1 η indicates the two enzymeshave similar reaction rate and product inhibition. hNPP1 has slightlyfaster rate and thus depletes substrate ATP slightly faster than hNPP3at low substrate concentration.

FIG. 2 illustrates a non-limiting purification profile of NPP3 fusionprotein through a Coomasie stained sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) gel, wherein the purified. NPP3 proteinis shown in relation to certain size markers.

FIG. 3 illustrates a non-limiting plasmid construct map of humanNPP121-NPP3-Fc in the plasmid, cloned using the indicated restrictionendonuclease sites.

FIG. 4 illustrates a non-limiting plasmid construct map of humanNPP121-NPP3-Fc in the plasmid pcDNA3, cloned using IN-FUSION®technology.

FIG. 5 illustrates a non-limiting plasmid construct map of humanNPP121-NPP3-Albumin in the plasmid pcDNA3.

BRIEF SUMMARY OF IRE INVENTION

The invention provides an isolated polypeptide, or a pharmaceutical saltor solvate thereof. The invention further provides a method of treatingor preventing a disease or disorder associated with pathologicalcalcification or pathological ossification in a subject in need thereof.The invention further provides a method of reducing or preventingvascular calcification in a subject with low plasma pyrophosphate (PPi)or high serum phosphate (Pi). The invention further provides a method oftreating of a subject having NPP I deficiency or NPP1-associateddisease. The invention further provides a kit comprising at least oneisolated polypeptide of the invention and instructions reciting the useof the at least one polypeptide for treating a disease or disorderassociated with pathological calcification or pathological ossificationin a subject in need thereof, optionally further comprising anapplicator.

In certain embodiments, the polypeptide of the invention has formula(I): EXPORT-PROTEIN-Z-DOMAIN-X-Y (I), wherein in (I): EXPORT is absent,or a signal export sequence or a biologically active fragment thereofPROTEIN is the extracellular domain of ENPP3 (SEQ ID NO:1) or abiologically active fragment thereof; DOMAIN is selected from the groupconsisting of a human IgG Fc domain and human albumin domain; X and Zare independently absent or a polypeptide comprising 1-20 amino acids;and, Y is absent or a sequence selected from the group consisting of:(DSS)_(n) (SEQ ID NO:6), (ESS)_(n) (SEQ ID NO:7), (RQQ)_(n) (SEQ IDNO:8), (KR)_(n) (SEQ ID NO:9), R_(n) (SEQ ID NO:10), (KR)_(n) (SEQ IDNO:11), DSSSEEKFLRRIGRFG (SEQ ID NO:12), EEEEEEEPRGDT (SEQ ID NO:13),APWHLSSQYSRT (SEQ ID NO:14), STLPIPHEFSRE (SEQ ID NO:15), VTKHLNQISQSY(SEQ ID NO:16), E_(n) (SEQ ID NO:17), and D_(n) (SEQ ID NO:18), whereineach occurrence of n is independently an integer ranging from 1 to 20.

In certain embodiments, the nuclease domain of the PROTEIN or mutantthereof is absent. In other embodiments. EXPORT is absent or selectedfrom the group consisting of SEQ ID NOs:2-5. In yet other embodiments, Xis selected from the group consisting of: absent, a polypeptideconsisting of 20 amino acids, a polypeptide consisting of 19 aminoacids, a polypeptide consisting of 18 amino acids, a polypeptideconsisting of 17 amino acids, a polypeptide consisting of 16 aminoacids, a polypeptide consisting of 15 amino acids, a polypeptideconsisting of 14 amino acids, a polypeptide consisting of 13 aminoacids, a polypeptide consisting of 12 amino acids, a polypeptideconsisting of 11 amino acids, a polypeptide consisting of 10 aminoacids, a polypeptide consisting of 9 amino acids, a polypeptideconsisting of 8 amino acids, a polypeptide consisting of 7 amino acids,a polypeptide consisting of 6 amino acids, a polypeptide consisting of 5amino acids, a polypeptide consisting of 4 amino acids, a polypeptideconsisting of 3 amino acids, a polypeptide consisting of 2 amino acids,and a polypeptide consisting of 1 amino acid. In yet other embodiments,Z is selected from the group consisting of: absent, a polypeptideconsisting of 20 amino acids, a polypeptide consisting of 19 aminoacids, a polypeptide consisting of 18 amino acids, a polypeptideconsisting of 17 amino acids, a polypeptide consisting of 16 aminoacids, a polypeptide consisting of 15 amino acids, a polypeptideconsisting of 14 amino acids, a polypeptide consisting of 13 aminoacids, a polypeptide consisting of 12 amino acids, a polypeptideconsisting of 11 amino acids, a polypeptide consisting of 10 aminoacids, a polypeptide consisting of 9 amino acids, a polypeptideconsisting of 8 amino acids, a polypeptide consisting of 7 amino acids,a polypeptide consisting of 6 amino acids, a polypeptide consisting of 5amino acids, a polypeptide consisting of 4 amino acids, a polypeptideconsisting of 3 amino acids, a polypeptide consisting of 2 amino acids,and a polypeptide consisting of 1 amino acid.

In certain embodiments, DOMAIN is a human IgG Fe domain selected fromthe group consisting of IgG1, IgG2, IgG3 and IgG4. In other embodiments,the polypeptide is selected from the group consisting of SEQ NOs:19, 21and 22. In yet other embodiments, DOMAIN is a human albumin domain. Inyet other embodiments, the polypeptide is selected from the groupconsisting of SEQ ID NOs:24, 25 and 26.

In certain embodiments, the polypeptide comprises a soluble region ofNPP3 and lacks a transmembrane domain and a signal peptide, or a fusionprotein thereof, wherein the polypeptide reduces cellular calcificationwhen administered to a subject suffering from diseases of calcificationand ossification. In other embodiments, the polypeptide comprises asoluble region of NPP3 and lacks a transmembrane domain and a signalpeptide, wherein the polypeptide reduces cellular calcification whenadministered to a subject suffering from diseases of calcification andossification.

In certain embodiments, the polypeptide comprises the extracellulardomain of ENPP3 (SEQ ID NO:1) or a biologically active fragment thereof.In other embodiments, the polypeptide consists essentially of SEQ IDNO:1 or a biologically active fragment thereof. In yet otherembodiments, the polypeptide consists of SEQ ID NO: 1 or a biologicallyactive fragment thereof.

In certain embodiments, the soluble ENPP3 fragment or fusion proteinthereof comprises the extracellular domain of ENPP3 (SEQ IL) NO:1) or abiologically active fragment thereof. In other embodiments, the solubleENPP3 fragment consists essentially of SEQ ID NO:1 or a biologicallyactive fragment thereof In yet other embodiments, the soluble ENPP3fragment consists of SEQ ID NO:1 or a biologically active fragmentthereof. In yet other embodiments, the soluble ENPP3 fragment or fusionprotein thereof lacks a transmembrane domain and a signal peptide.

In certain embodiments, the method comprises administering to thesubject a therapeutically effective amount of at least one polypeptidethe invention, or a pharmaceutical salt or solvate thereof. In otherembodiments, the method comprises administering to the subject atherapeutically effective amount of an isolated recombinant humansoluble ENPP3 fragment or fusion protein thereof.

In certain embodiments, the disease or disorder comprises at least oneselected from the group consisting of GACI, IIAC, PXE, OPLL,hypophosphatemic rickets, osteoarthritis, calcification ofatherosclerotic plaques, hereditary and non-hereditary forms ofosteoarthritis, ankylosing spondylitis, hardening of the arteriesoccurring with aging, and calciphylaxis resulting from end stage renaldisease (or mineral bone disorder of chronic kidney disease).

In certain embodiments, the disease or disorder comprises at least oneselected from a group consisting of GACI, IIAC, PXE, OPLL, MWVC, ARHR2,ESRD, CKD-MBD, XLH, age related osteopenia, CUA and hypophosphatemicrickets.

In certain embodiments, the disease or disorder is GACI. In otherembodiments, the disease or disorder is IIAC. In yet other embodiments,the disease or disorder is PXE. In vet other embodiments, the disease ordisorder is OPLL. In yet other embodiments, the disease or disorder ishypophosphatemic rickets. In yet other embodiments, the disease ordisorder is osteoarthritis. In vet other embodiments, the disease ordisorder is calcification of atherosclerotic plaques. In yet otherembodiments, the disease or disorder is hereditary and non-hereditaryforms of osteoarthritis. In yet other embodiments, the disease ordisorder is ankylosing spondylitis. In vet other embodiments, thedisease or disorder is hardening of the arteries occurring with aging.In yet other embodiments, the disease or disorder is calciphylaxisresulting from end stage renal disease (or mineral bone disorder ofchronic kidney disease). In yet other embodiments, the disease ordisorder is age related osteopenia. In yet other embodiments, thedisease or disorder is CUA. In yet other embodiments, the disease ordisorder is MWVC. In yet other embodiments, the disease or disorder isARHR2. In yet other embodiments, the disease or disorder is ESRD.

In certain embodiments, the administered amount raises the level ofplasma PPi in the subject to at least about 800 nM. In otherembodiments, the administered amount raises the level of plasma PPi inthe subject to at least about 1 μM. In yet other embodiments, theadministered amount raises the level of plasma PPi in the subject to atleast about 1.5 μM.

In certain embodiments, the at least one polypeptide is administeredacutely or chronically to the subject. In other embodiments, the atleast one polypeptide is administered locally, regionally orsystemically to the subject. In yet other embodiments, the subject is amammal. In yet other embodiments, the mammal is human.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that ENPP3 (also known asNPP3), which is a member of the ectonucleotidepyrophosphatase/phosphodiesterase (ENPP or NPP) family of enzymes, haspotent ATP hydrolase activity. ENPP3 hydrolyzes ATP to AMP and PPi, asdemonstrated herein.

In certain aspects, the present invention provides compositions, such asbut not limited to fusion proteins, that elevate plasma PPi inphysiologic states where plasma PPi is low (as determined, for example,by a medical professional or by consulting of a medical document ormanual), placing the individual at risk of morbidity associated with lowPPi states. In certain embodiments, these physiologic states arerecognized disease conditions such as GACI, PXE, Hutchinson GilfordProgeria Syndrome, chronic kidney disease (CKD), X-linkedhypophosphatemia, sickle cell anemia, and end stage renal disease. Inother embodiments, these physiologic states occur in non-disease states,such as in elderly adults who are afflicted with chronic ailments knownto occur in all aging adults such as “hardening of the arteries” andosteopenia.

In certain embodiments, low plasma PPi is defined as plasma PPiconcentration lower than about 1.5 μM. These disease states may or maynot be accompanied by pathologic calcification of the arteries and/orsoft tissues, medial vascular wall calcifications, strokes orcerebrovascular accidents, decreased pulse wave velocity, calcificationsof the soft tissues such as the skin, calcifications of the Bruchsmembrane in the eye, calcifications of soft tissues surrounding tendonsalso known as entheses, calcifications of ligaments in the spine such asthe posterior longitudinal ligament, and disease of ossification such asRickets. In other embodiments, the invention contemplates treatment oflow PPi physiologic states via administration of the fusion proteinsdescribed herein.

In other aspects, the compositions and methods of the invention can beused to treat disease states known to occur in conditions where theexpression or the activity of the enzyme ENPP1 is reduced. Theserecognized disease states include, in non-limited manner,osteoarthritis, GACI, and ARHR2. These states may also occur in otherphysiologic states in which ENPP1 protein levels are reduced, such as inindividuals who have a common polymporphism in the ENPP1 coding regionin which a Q residue is substituted for a K reside at position 121 ofthe secreted protein (or position 173 of the full length protein)(Eller, et al., 2008, Nephrol. Dial. Transplant. 23(1):321-7; Flanagan,et al., 2013, Blood 121(16):3237-45).

As demonstrated herein, the products of ATP hydrolysis by ENPP3, and thecorresponding enzymatic constants, were analyzed in order to study theenzymatic activity of this enzyme. ENPP3 was found to he a potent _ATPhydrolase, capable of generating PPi and AMP from ATP. In certainembodiments, ENPP3 has an ATP hydrolase activity that is comparable tothat of ENPP1. As demonstrated herein, ENPP3 catalyzes the hydrolysis ofATP to PPi with nearly the same Michaelis-Menton kinetics as ENPP1,which is another member of the ENPP family of enzymes. In certainembodiments, soluble fusion constructs of ENPP3, including albuminfusion constructs thereof and/or IgG Fc domain constructs thereof, areefficacious in treating diseases of ectopic calcification. In yet otherembodiments, the constructs described herein are efficacious in treatingand/or preventing disorders of ectopic vascular calcification.

In one aspect NPP3 is poorly exported to the cell surface. In certainembodiments, soluble ENPP3 protein is constructed by replacing thesignal sequence of NPP3 with the native signal sequence of other ENPPs.In other embodiments, soluble ENPP3 constructs are prepared by using thesignal export signal sequence of other ENPP enzymes, such as but notlimited to ENPP7 and/or ENPPS. In yet other embodiments, soluble ENPP3constructs are prepared by using a signal sequence comprised of acombination of the signal sequences of ENPP1 and ENPP2 (“ENPP1-2-1”hereinafter). In yet other embodiments, signal sequences of any otherknown proteins may be used to target the extracellular domain of ENPP3for secretion as well, such as but not limited to the signal sequence ofthe immunoglobulin kappa and lambda light chain proteins. Further, theinvention should not be construed to be limited to the constructsdescribed herein, but also includes constructs comprising anyenzymatically active truncation of the ENPP3 extracellular domain.

Diseases and disorders involving pathological calcification and/orpathological ossification treatable by the compositions and methods ofthe invention, include, but are not limited to, Idiopathic InfantileArterial Calcification (IIAC), Ossification of the PosteriorLongitudinal Ligament (OPLL), hypophosphatemic rickets, osteoarthritis,calcification of atherosclerotic plaques, Pseudoxanthoma elasticum(PXE), hereditary and non-hereditary forms of osteoarthritis, ankylosingspondylitis, hardening of the arteries occurring with aging, andcalciphylaxis resulting from end stage renal disease.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

As used herein, the term “AMR” refers to autosomal dominanthypophosphatemic rickets.

As used herein, the term “albumin” refers to the blood plasma proteinthat is produced in the liver and forms a large proportion of all plasmaprotein. In certain embodiments, albumin refers to human serum albumin.Usage of other albumins such as bovine serum albumin, equine serum albumand porcine serum albumin are also contemplated within the invention.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

As used herein the terms “alteration,” “defect,” “variation” or“mutation” refer to a mutation in a gene in a cell that affects thefunction, activity, expression (transcription or translation) orconformation of the polypeptide it encodes. Mutations encompassed by thepresent invention can be any mutation of a gene in a cell that resultsin the enhancement or disruption of the function, activity, expressionor conformation of the encoded polypeptide, including the completeabsence of expression of the encoded protein and can include, forexample, missense and nonsense mutations, insertions, deletions,frameshifts and premature terminations. Without being so limited,mutations encompassed by the present invention may alter splicing themRNA (splice site mutation) or cause a shift in the reading frame(frameshift).

The term “amino acid sequence variant” refers to polypeptides havingamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants possess at leastabout 70% homology, at least about 80% homology, at least about 90%homology, or at least about 95% homology to the native polypeptide. Theamino acid sequence variants possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence of thenative amino acid sequence.

As used herein, the term “Ap3P” refers toadenosine-(5′)-triphospho-(5′)-adenosine or a salt thereof.

As used herein, the term “ARHR2” refers to autosomal recessivehypophosphatemic rickets type-2.

As used herein, the term “CKD” refers to chronic kidney disease.

As used herein, the term “CKD-MBD” refers to chronic kidneydisease-bone/mineral disorder.

The term “coding sequence,” as used herein, means a sequence of anucleic acid or its complement, or a part thereof that can betranscribed and/or translated to produce the mRNA and/or the polypeptideor a fragment thereof. Coding sequences include exons in a genomic DNAor immature primary RNA transcripts, which are joined together by thecell's biochemical machinery to provide a mature mRNA. The anti-sensestrand is the complement of such a nucleic acid, and the coding sequencecan be deduced therefrom. In contrast, the term “non-coding sequence,”as used herein, means a sequence of a nucleic acid or its complement, ora part thereof, that is not translated into amino acid in vivo, or wheretRNA does not interact to place or attempt to place an amino acid.Non-coding sequences include both intron sequences in genomic DNA orimmature primary RNA transcripts, and gene-associated sequences such aspromoters, enhancers, silencers, and the like.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. Or, there may be “complete” or “total”complementarity between the nucleic acids. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, as well as detectionmethods that depend upon binding between nucleic acids.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a patient.Multiple techniques of administering a compound exist in the artincluding, but not limited to, intravenous, oral, aerosol, inhalational,rectal, vaginal, transdermal, intranasal, buccal, sublingual,parenteral, intrathecal, intragastrical, ophthalmic, pulmonary andtopical administration.

As used herein, the terms “conservative variation” or “conservativesubstitution” as used herein refers to the replacement of an amino acidresidue by another, biologically similar residue. Conservativevariations or substitutions are not likely to change the shape of thepeptide chain. Examples of conservative variations, or substitutions,include the replacement of one hydrophobic residue such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acid, or glutamine for asparagine.

As used herein, the term “CUA” refers to calcific uremic arteriolopaihy.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

A “disorder” in an animal is a state of health in which the animal isable to maintain homeostasis, but in which the animal's state of healthis less favorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

As used herein, the term “domain” refers to a part of a molecule orstructure that shares common physicochemical features, such as, but notlimited to, hydrophobic, polar, globular and helical domains orproperties. Specific examples of binding domains include, but are notlimited to, DNA binding domains and ATP binding domains.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. An appropriate therapeutic amount inany individual ease may be determined by one of ordinary skill in theart using routine experimentation.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein, the term “ESRD” refers to end-stage renal disease. Asused herein, the term “Fc” refers to a human IgG Fc domain. Subtypes ofIgG such as IgG1, IgG2, IgG3, and IgG4 are all being contemplated forusage as Fc domains.

As used herein, the term “fragment,” as applied to a nucleic acid,refers to a subsequence of a larger nucleic acid. A “fragment” of anucleic acid can be at least about 15 nucleotides in length; forexample, at least about 50 nucleotides to about 100 nucleotides; atleast about 100 to about 500 nucleotides, at least about 500 to about1000 nucleotides; at least about 1000 nucleotides to about 1500nucleotides; about 1500 nucleotides to about 2500 nucleotides; or about2500 nucleotides (and any integer value in between). As used herein, theterm “fragment,” as applied to a protein or peptide, refers to asubsequence of a larger protein or peptide. A “fragment” of a protein orpeptide can be at least about 20 amino acids in length; for example, atleast about 50 amino acids in length; at least about 100 amino acids inlength; at least about 200 amino acids in length; at least about 300amino acids in length; or at least about 400 amino acids in length (andany integer value in between).

As used herein, the term “HBD” refers to hypophosphatemic bone disease.

As used herein, the term “HHRH” refers to hereditary hypophosphatemicrickets with hypercakiuria.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared X 100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

As used herein, the term “IIAC” refers to idiopathic infantile arterialcalcification.

As used herein, an “immunoassay” refers to any binding assay that usesan antibody capable of binding specifically to a target molecule todetect and quantify the target molecule.

As used herein, the term “immunoglobulin” or “Ig” is defined as a classof proteins that function as antibodies. Antibodies expressed by B cellsare sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgS9, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody 15 responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the nucleic acid,peptide, and/or compound of the invention in the kit for identifying oralleviating or treating the various diseases or disorders recitedherein. Optionally, or alternately, the instructional material maydescribe one or more methods of identifying or alleviating the diseasesor disorders in a cell or a tissue of a subject. The instructionalmaterial of the kit may, for example, be affixed to a container thatcontains the nucleic acid, polypeptide, and/or compound of the inventionor be shipped together with a container that contains the nucleic acid,polypeptide, and/or compound. Alternatively, the instructional materialmay be shipped separately from the container with the intention that therecipient uses the instructional material and the compoundcooperatively. Alternatively, the kit comprises an applicator that canbe used to administer the nucleic acid, peptide, and/or compound of theinvention to the subject. The application may be for example a dropdispenser, a bottle, a pill dispenser, a syringe and so forth.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a polypeptide naturally present in a living animal isnot “isolated,” but the same nucleic acid or polypeptide partially orcompletely separated from the coexisting materials of its natural stateis “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

As used herein, the term “MWVC” refers to medial wall vascularcalcification.

As used herein, the term “NPP” refers to ectonucleotidepyrophosphatase/phosphodiesterase.

A “nucleic acid” refers to a polynucleotide and includespoly-ribonucleotides and poly-deoxyribonucleotides. Nucleic acidsaccording to the present invention may include any polymer or oligomerof pyrimidine and purine bases, preferably cytosine, thymine, anduracil, and adenine and guanine, respectively (Lehninger, Principles ofBiochemistry, at 793-800 (Worth Pub. 1982), which is herein incorporatedin its entirety for all purposes). Indeed, the present inventioncontemplates any deoxyribonucleotide, ribonucleotide or peptide nucleicacid component, and any chemical variants thereof, such as methylated,hydroxymethylated or glucosylated forms of these bases, and the like.The polymers or oligomers may be heterogeneous or homogeneous incomposition, and may be isolated from naturally occurring sources or maybe artificially or synthetically produced. In addition, the nucleicacids may be DNA or RNA, or a mixture thereof, and may exist permanentlyor transitionally in single-stranded or double-stranded form, includinghomoduplex, heteroduplex, and hybrid states.

An “oligonucleotide” or “polynucleotide” is a nucleic acid ranging fromat least 2, preferably at least 8, 15 or 25 nucleotides in length, butmay be up to 50, 100, 1000, or 5000 nucleotides long or a compound thatspecifically hybridizes to a polynucleotide. Polynucleotides includesequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) ormimetics thereof which may be isolated from natural sources,recombinantly produced or artificially synthesized. A further example ofa polynucleotide of the present invention may be a peptide nucleic acid(PNA). (See U.S. Pat. No. 6,156,501 which is hereby incorporated byreference in its entirety) The invention also encompasses situations inwhich there is a nontraditional base pairing such as Hoogsteen basepairing which has been identified in certain tRNA molecules andpostulated to exist in a triple helix. “Polynucleotide” and“oligonucleotide” are used interchangeably herein. When a nucleotidesequence is represented herein by a DNA sequence (e.g., A, T, G, and C),this also includes the corresponding RNA sequence (e.g., A, U, G, C) inwhich “U” replaces “T.”

As used herein, the term “OPLL” refers to ossification of posteriorlongitudinal ligament.

As used herein, the term “patient,” “individual” or “subject” refers toa human or a non-human mammal. Non-human mammals include, for example,livestock and pets, such as ovine, bovine, porcine, canine, feline andmurine mammals. Exemplarily, the patient, individual or subject ishuman.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate, and ethyllaurate; agar; buffering agents, such as magnesium hydroxide andaluminum hydroxide; surface active agents; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations. “Pharmaceutically acceptable carrier” alsoincludes any and all coatings, antibacterial and antifungal agents, andabsorption delaying agents, and the like that are compatible with theactivity of the compound useful within the invention, and arephysiologically acceptable to the patient. Supplementary activecompounds may also be incorporated into the compositions. The“pharmaceutically acceptable carrier” may further include apharmaceutically acceptable salt of the compound useful within theinvention. Other additional ingredients that may he included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compound prepared from pharmaceuticallyacceptable non-toxic acids and bases, including inorganic acids,inorganic bases, organic acids, inorganic bases, solvates, hydrates, andclathrates thereof. Suitable pharmaceutically acceptable acid additionsalts may be prepared from an inorganic acid or from an organic acid.Examples of inorganic acids include sulfate, hydrogen sulfate,hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, andphosphoric acids (including hydrogen phosphate and dihydrogenphosphate). Appropriate organic acids may be selected from aliphatic,cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic andsulfonic classes of organic acids, examples of which include formic,acetic, propionic, succinic, glycolic gluconic, lactic, malic, tartaric,citric, ascorbic, glucuronic, maleic, fumaric, pymvic, aspartic,glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic,mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic,stearic, alginic, β-hydroxy butyric, salicylic, galactaric andgalacturonic acid. Suitable pharmaceutically acceptable base additionsalts of compounds of the invention include, for example, metallic saltsincluding alkali metal, alkaline earth metal and transition metal saltssuch as, for example, calcium, magnesium, potassium, sodium and zincsalts. Pharmaceutically acceptable base addition salts also includeorganic salts made from basic amines such as, for example,N,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. All ofthese salts may be prepared from the corresponding compound by reacting,for example, the appropriate acid or base with the compound.

As used herein, the term “plasma pyrophosphate levels” or “plasma PPi”refers to the amount of pyrophosphate (PPi) present in plasma ofanimals. In certain embodiments, animals include mammals, such as butnot limited to rat, mouse, cat, dog, human, cow and horse. In certainembodiments, PPi is measured in plasma rather than serum, because of itsrelease from platelets. There are several non-limiting ways to measurePPi, one of which is by enzymatic assay using uridine-diphosphoglucose(UDPG) pyrophosphorylase as described by Lust and Seegmiller (Lust, etal., 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977. Anal.Biochem. 83:61-63) with modifications. Typically healthy individualsexhibit a mean plasma level of about 3.0 μM. The levels of plasma PPi insubjects with aging and or with diseases of calcification orossification are much lower than the normal levels. In certainembodiments, subjects exhibit a low plasma PPi level of about 1.5 μm. Inother embodiments, for subjects with diseases of calcification theplasma PPi levels are about 500 nM, about 600 nM, about 700 nM, about800 nM, about 900 nM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8μM, about 1.9 μM, about 2 μM about 2.2 μM, about 2.4 μM, and/or about2.6 μM. In yet other embodiments, for subjects with diseases ofcalcification the plasma PPi levels range from about 500 nM to about 2.8μM, about 600 nM to about 2.8 μM, about 700 nM to about 2.8 μM, about800 nM to about 2.8 μM, about 900 nM to about 2.8 μM, about 1 μM toabout 2.8 μM, about 1.1 μM to about 2.8 μM, about 1.2 μM to about 2.8μM, about 1.3 μM to about 2.8 μM, about 1.4 μM to about 2.8 μM, about1.5 μM to about 2.8 μM, about 1.6 μM to about 2.8 μM, about 1.7 μM toabout 2.8 μM, about 1.8 μM to about 2.8 μM, about 1.9 μM to about 2.8μM, about 2 μM to about 2.8 μM, about 2.2 μM to about 2.8 μM about 2.4μM to about 2.8 μM, and/or about 2.6 μM to about 2.8 μM.

As used herein, “polynucleotide” includes cDNA, RNA, DNA/RNA hybrid,antisense RNA, ribozyme, genomic DNA, synthetic forms, and mixedpolymers, both sense and antisense strands, and may be chemically orbiochemically modified to contain non-natural or derivatized, synthetic,or semi-synthetic nucleotide bases. Also, contemplated are alterationsof a wild type or synthetic gene, including but not limited to deletion,insertion, substitution of one or more nucleotides, or fusion to otherpolynucleotide sequences.

As used herein, the term “polypeptide” refers to a polymer composed ofamino acid residues, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof linked via peptidebonds. Synthetic polypeptides may be synthesized, for example, using anautomated polypeptide synthesizer. As used herein, the term “protein”typically refers to large polypeptides. As used herein, the term“peptide” typically refers to short polypeptides. Conventional notationis used herein to represent polypeptide sequences: the left-hand end ofa polypeptide sequence is the amino-terminus, and the right-hand end ofa polypeptide sequence is the carboxyl-terminus.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated below: Aspartic Acid, Asp, D;Glutamic Acid, (Hu, B; Lysine, Lys, K; Arginine, Arg, R; Histidine, His,H; Tyrosine, Tyr, Y; Cysteine, Cys, C; Asparagine, Asn, N; Glutamine,Gln, Q; Serine, Ser, S; Threonine, Thr, T; Glycine, Gly, G; Alanine,Ala, A; Valine, Val, V; Leucine, Leu, L; Isoleucine, Ile, I; Methionine,Met, M; Proline, Pro, P; Phenylalanine, Phe, F; Tryptophan, W.

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.

As used herein, the term “PXE” refers to pseudoxanthoma elasticum.

“Sample” or “biological sample” as used herein means a biologicalmaterial isolated from a subject. The biological sample may contain anybiological material suitable for detecting a mRNA, polypeptide or othermarker of a physiologic or pathologic process in a subject, and maycomprise fluid, tissue, cellular and/or non-cellular material obtainedfrom the individual.

As used herein, “substantially purified” refers to being essentiallyfree of other components. For example, a substantially purifiedpolypeptide is a polypeptide which has been separated from othercomponents with which it is normally associated in its naturallyoccurring state.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compounduseful within the invention (alone or in combination with anotherpharmaceutical agent), to a patient, or application or administration ofa therapeutic agent to an isolated tissue or cell line from a patient(e.g., for diagnosis or ex vivo applications), who has a disease ordisorder, a symptom of a disease or disorder or the potential to developa disease or disorder, with the purpose to cure, heal, alleviate,relieve, alter, remedy, ameliorate, improve or affect the disease ordisorder, the symptoms of the disease or disorder, or the potential todevelop the disease or disorder. Such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics.

As used herein, the term “XLH” refers to X-linked hypophosphatemia,X-linked dominant hypophosphatemic rickets, X-linked vitamin D-resistantrickets, and/or X-linked hypophosphatemic rickets.

As used herein, the term “wild-type” refers to a gene or gene productisolated from a naturally occurring source. A wild-type gene is thatwhich is most frequently observed in a population and is thusarbitrarily designed the “normal” or “wild-type” form of the gene. Incontrast, the term “modified” or “mutant” refers to a gene or geneproduct that displays modifications in sequence and/or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene or gene product. Naturally occurring mutants can beisolated; these are identified by the fact that they have alteredcharacteristics (including altered nucleic acid sequences) when comparedto the wild-type gene or gene product.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Compositions

In certain embodiments, the polypeptide of the invention has formula(I): EXPORT-PROTEIN-Z-DOMAIN-X-Y (I), wherein in (I): EXPORT is absent,or a signal export sequence or a biologically active fragment thereof;PROTEIN is the extracellular domain of ENPP3 (SEQ IL) NO:1) or abiologically active fragment thereof; DOMAIN is selected from the groupconsisting of a human IgG Fc domain and human albumin domain; X and Zare independently absent or a polypeptide comprising 1-20 amino acids;and, Y is absent or a sequence selected from the group consisting of(DSS)_(n)(SEQ ID NO:6), (ESS)_(n) (SEQ Ill NO:7), (RQQ)_(n) (SEQ NO:8),(KR)_(n) (SEQ NO:9), R_(n) (SEQ ID NO:10), (KR)_(n) (SEQ ID NO: 11),DSSSEEKFLRRIGRFG (SEQ ID NO:12), EEEEEEEPRGDT (SEQ ID NO:13),APWHLSSQYSRT (SEQ ID NO:14), STLPIPHEFSRE (SEQ ID NO:15), VTKHLNQISQSY(SEQ ID NO:16), E_(n) (SEQ ID NO:17), and D_(n) (SEQ ID NO:18), whereineach occurrence of n is independently an integer ranging from 1 to 20.

In certain embodiments, the polypeptide comprises the extracellulardomain of ENPP3 (SEQ ID NO:1) or a biologically active fragment (orregion) thereof.

In certain embodiments, the polypeptide is soluble. In otherembodiments, the nuclease domain of the PROTEIN or mutant thereof isabsent. In yet other embodiments, EXPORT is absent or selected from thegroup consisting of SEQ ID NOs:2-5. In yet other embodiments, X isselected from the group consisting of: absent, a polypeptide consistingof 20 amino acids, a polypeptide consisting of 19 amino acids, apolypeptide consisting of 18 amino acids, a polypeptide consisting of 17amino acids, a polypeptide consisting of 16 amino acids, a polypeptideconsisting of 15 amino acids, a polypeptide consisting of 14 aminoacids, a polypeptide consisting of 13 amino acids, a polypeptideconsisting of 12 amino acids, a polypeptide consisting of 11 aminoacids, a polypeptide consisting of 10 amino acids, a polypeptideconsisting of 9 amino acids, a polypeptide consisting of 8 amino acids,a polypeptide consisting of 7 amino acids, a polypeptide consisting of 6amino acids, a polypeptide consisting of 5 amino acids, a polypeptideconsisting of 4 amino acids, a polypeptide consisting of 3 amino acids,a polypeptide consisting of 2 amino acids, and a polypeptide consistingof 1 amino acid. In vet other embodiments, Z is selected from the groupconsisting of: absent, a polypeptide consisting of 20 amino acids, apolypeptide consisting of 19 amino acids, a polypeptide consisting of 18amino acids, a polypeptide consisting of 17 amino acids, a polypeptideconsisting of 16 amino acids, a polypeptide consisting of 15 aminoacids, a polypeptide consisting of 14 amino acids, a polypeptideconsisting of 13 amino acids, a polypeptide consisting of 12 aminoacids, a polypeptide consisting of 11 amino acids, a polypeptideconsisting of 10 amino acids, a polypeptide consisting of 9 amino acids,a polypeptide consisting of 8 amino acids, a polypeptide consisting of 7amino acids, a polypeptide consisting of 6 amino acids, a polypeptideconsisting of 5 amino acids, a polypeptide consisting of 4 amino acids,a polypeptide consisting of 3 amino acids, a polypeptide consisting of 2amino acids, and a polypeptide consisting of 1 amino acid.

In certain embodiments, X and Z are independently absent or apolypeptide comprising 1-18 amino acids. In other embodiments, X and Zare independently absent or a polypeptide comprising 1-16 amino acids.In yet other embodiments, X and Z are independently absent or apolypeptide comprising 1-14 amino acids. In yet other embodiments, X andZ are independently absent or a polypeptide comprising 1-12 amino acids.In yet other embodiments, X and Z are independently absent or apolypeptide comprising 1-10 amino acids. In yet other embodiments, X andZ are independently absent or a polypeptide comprising 1-8 amino acids.In yet other embodiments, X and Z are independently absent or apolypeptide comprising 1-6 amino acids. In yet other embodiments, X andZ are independently absent or a polypeptide comprising 1-5 amino acids.In yet other embodiments, X and Z are independently absent or apolypeptide comprising 1-4 amino acids, In yet other embodiments, X andZ are independently absent or a polypeptide comprising 1-3 amino acids.In yet other embodiments, X and Z are independently absent or apolypeptide comprising 1-2 amino acids. In yet other embodiments, X andZ are independently absent or a single amino acid.

In certain embodiments, DOMAIN is a human IgG Fe domain selected fromthe group consisting of IgG 1, IgG2, IgG3 and IgG4. In otherembodiments, the polypeptide is selected from the group consisting ofSEQ ID NOs: 19, 21 and 22. In yet other embodiments, DOMAIN is a humanalbumin domain. In vet other embodiments, the polypeptide is selectedfrom the group consisting of SEQ ID NOs: 24, 25 and 26.

In certain embodiments, the soluble polypeptide lacks a transmembranedomain and/or signal peptide. In other embodiments, the solublepolypeptide lacks a transmembrane domain. In yet other embodiments, thesoluble polypeptide lacks a signal peptide. In yet other embodiments,the soluble polypeptide lacks a transmembrane domain and signal peptide.

In certain embodiments, the polypeptide comprises a soluble region (orfragment) of NPP3 and lacks a transmembrane domain and a signal peptide,or a fusion protein thereof. In other embodiments, the polypeptidecomprises a soluble region of NPP3 and lacks a transmembrane domainand/or a signal peptide. In yet other embodiments, the polypeptidecomprises a soluble region of NPP3 and lacks a transmembrane domain. Inyet other embodiments, the polypeptide comprises a soluble region ofNPP3 and lacks a signal peptide. In yet other embodiments, thepolypeptide reduces cellular calcification when administered to asubject suffering from diseases of calcification and ossification.

In certain embodiments, the polypeptide consists essentially of SEQ IDNO:1 or a biologically active fragment thereof. In other embodiments,the polypeptide consists of SEQ ID NO:1 or a biologically activefragment thereof.

In certain embodiments, the soluble ENPP3 fragment or fusion proteinthereof comprises the extracellular domain of ENPP3 (SEQ ID NO:1) or abiologically active fragment thereof. In other embodiments, the solubleENPP3 fragment consists essentially of SEQ ID NO:1 or a biologicallyactive fragment thereof In yet other embodiments, the soluble ENPP3fragment consists of SEQ ID NO:1 or a biologically active fragmentthereof. In yet other embodiments, the soluble ENPP3 fragment or fusionprotein thereof lacks a transmembrane domain and a signal peptide.

In certain embodiments, the polypeptide of the invention is soluble. Inother embodiments, the polypeptide of the invention is a recombinantpolypeptide. In yet other embodiments, the polypeptide of the inventionis further pegylated.

Methods

The invention provides a method of treating or preventing a disease ordisorder associated with pathological calcification or pathologicalossification in a subject in need thereof. The invention furtherprovides a method of reducing or preventing vascular calcification in asubject with low plasma pyrophosphate (PPi) or high serum phosphate(Pi). The invention further provides a method of treating of a subjecthaving NPP1. deficiency or NPP1-associated disease. The inventionfurther provides a method of treating or preventing disorders anddiseases in a subject where an increased activity or level of ENPP3polypeptide, fragment, derivative, mutant, or mutant fragment thereof isdesirable.

In certain embodiments, the subject is administered a therapeuticallyeffective amount of at least one polypeptide of the invention. In otherembodiments, the method comprises administering to the subject atherapeutically effective amount of an isolated recombinant humansoluble ENPP3 fragment or fusion protein thereof.

In certain embodiments, the disease or disorder comprises at least oneselected from the group consisting of GACI, IIAC, PXE, OPLL,hypophosphatemic rickets, osteoarthritis, calcification ofatherosclerotic plaques, hereditary and non-hereditary forms ofosteoarthritis, ankylosing spondylitis, hardening of the arteriesoccurring with aging, and calciphylaxis resulting from end stage renaldisease (or mineral bone disorder of chronic kidney disease).

In certain embodiments, the disease or disorder comprises at least oneselected from a group consisting of GACI, IIAC, PXE, OPLL, MWVC, ARHR2,ESRD, CKD-MBD, XLH, age related osteopenia, CUA and hypophosphatemicrickets.

In certain embodiments, the disease or disorder is GACI. In otherembodiments, the disease or disorder is IIAC. In yet other embodiments,the disease or disorder is PXE. In yet other embodiments, the disease ordisorder is OPLL. In yet other embodiments, the disease or disorder ishypophosphatemic rickets. In yet other embodiments, the disease ordisorder is osteoarthritis. In yet other embodiments, the disease ordisorder is calcification of atherosclerotic plaques. In yet otherembodiments, the disease or disorder is hereditary and non-hereditaryforms of osteoarthritis. In yet other embodiments, the disease ordisorder is ankylosing spondylitis. In yet other embodiments, thedisease or disorder is hardening of the arteries occurring with aging.In yet other embodiments, the disease or disorder is calciphylaxisresulting from end stage renal disease (or mineral bone disorder ofchronic kidney disease). In yet other embodiments, the disease ordisorder is age related osteopenia. In yet other embodiments, thedisease or disorder is CUA. In yet other embodiments, the disease ordisorder is MWVC. In yet other embodiments, the disease or disorder isARHR2. In yet other embodiments, the disease or disorder is ESRD.

In certain embodiments, the at least one polypeptide is administeredacutely or chronically to the subject. In other embodiments, the atleast one polypeptide is administered locally, regionally orsystemically to the subject. In yet other embodiments, the subject is amammal. In yet other embodiments, the mammal is human.

In certain embodiments, the administered amount raises the level ofplasma PPi in the subject to at least about 250 nM. In otherembodiments, the administered amount raises the level of plasma PPi inthe subject to at least about 500 nM. In yet other embodiments, theadministered amount raises the level of plasma PPi in the subject to atleast about 800 nM. In yet other embodiments, the administered amountraises the level of plasma PPi in the subject to at least about 900 nM,in yet other embodiments, the administered amount raises the level ofplasma PPi in the subject to at least about 1 μM. In yet otherembodiments, the administered amount raises the level of plasma PPi inthe subject to at least about 1.2 μM. In yet other embodiments, theadministered amount raises the level of plasma PPi in the subject to atleast about 1.4 μM. In yet other embodiments, the administered amountraises the level of plasma PPi in the subject to at least about 1.5 μM.In certain embodiments, the administered amount raises the level ofplasma PPi in the subject to at least about 2 μM. In certainembodiments, the administered amount raises the level of plasma PPi inthe subject to at least about 4 μM.

One skilled in the art, based upon the disclosure provided herein, wouldunderstand that the invention is useful in subjects who, in whole (e.g.,systemically) or in part (e.g., locally, tissue, organ), are being, orwill be, treated for pathological calcification or ossification. Incertain embodiments, the invention is useful in treating or preventingpathological calcification or ossification. The skilled artisan willappreciate, based upon the teachings provided herein, that the diseasesand disorders treatable by the compositions and methods described hereinencompass any disease or disorder where a decrease in calcification orossification will promote a positive therapeutic outcome.

It will be appreciated by one of skill in the art, when armed with thepresent disclosure including the methods detailed herein, that theinvention is not limited to treatment of a disease or disorder once isestablished. Particularly, the symptoms of the disease or disorder neednot have manifested to the point of detriment to the subject; indeed,the disease or disorder need not be detected in a subject beforetreatment is administered. That is, significant pathology from diseaseor disorder does not have to occur before the present invention mayprovide benefit. Therefore, the present invention, as described morefully herein, includes a method for preventing diseases and disorders ina subject, in that a polypeptide of the invention, or a mutant thereof,as discussed elsewhere herein, can be administered to a subject prior tothe onset of the disease or disorder, thereby preventing the disease ordisorder from developing.

One of skill in the art, when armed with the disclosure herein, wouldappreciate that the prevention of a disease or disorder in a subjectencompasses administering to a subject a polypeptide of the invention,or a mutant thereof as a preventative measure against a disease ordisorder.

The invention encompasses administration of a polypeptide of theinvention, or a mutant thereof to practice the methods of the invention;the skilled artisan would understand, based on the disclosure providedherein, how to formulate and administer the polypeptide of theinvention, or a mutant thereof to a subject. However, the presentinvention is not limited to any particular method of administration ortreatment regimen. This is especially true where it would be appreciatedby one skilled in the art, equipped with the disclosure provided herein,including the reduction to practice using an art-recognized model ofpathological calcification or ossification, that methods ofadministering a compound of the invention can be determined by one ofskill in the pharmacological arts.

Pharmaceutical Compositions and Formulations

The invention envisions the use of a pharmaceutical compositioncomprising a polypeptide of the invention within the methods of theinvention.

Such a pharmaceutical composition is in a form suitable foradministration to a subject, or the pharmaceutical composition mayfurther comprise one or more pharmaceutically acceptable carriers, oneor more additional ingredients, or some combination of these. Thevarious components of the pharmaceutical composition may be present inthe form of a physiologically acceptable salt, such as in combinationwith a physiologically acceptable cation or anion, as is well known inthe art.

In certain embodiments, the pharmaceutical compositions useful forpracticing the method of the invention may be administered to deliver adose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, thepharmaceutical compositions useful for practicing the invention may beadministered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between about 0.1% and about 100%(w/w) active ingredient.

Pharmaceutical compositions that are useful in the methods of theinvention may be suitably developed for inhalational, oral, rectal,vaginal, parenteral, topical, transdermal, pulmonary, intranasal,buccal, ophthalmic, intrathecal, intravenous or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations. Theroutes) of administration is readily apparent to the skilled artisan anddepends upon any number of factors including the type and severity ofthe disease being treated, the type and age of the veterinary or humanpatient being treated, and the like.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit. As used herein, a “unit dose” is a discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient that would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage. The unit dosage formmay be for a single daily dose or one of multiple daily doses (e.g.,about 1 to 4 or more times per day). When multiple daily doses are used,the unit dosage form may be the same or different for each dose.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions suitable forethical administration to humans, it is understood by the skilledartisan that such compositions are generally suitable for administrationo animals of all sorts. Modification of pharmaceutical compositionssuitable for administration to humans in order to render thecompositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

In certain embodiments, the compositions are formulated using one ormore pharmaceutically acceptable excipients or carriers. In certainembodiments, the pharmaceutical compositions comprise a therapeuticallyeffective amount of the active agent and a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers, which are useful,include, but are not limited to, glycerol, water, saline, ethanol andother pharmaceutically acceptable salt solutions such as phosphates andsalts of organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's PharmaceuticalSciences, 1991, Mack Publication Co., New Jersey.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,sodium chloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” that may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of exposure to contaminants in theenvironment. Examples of preservatives useful in accordance with theinvention included but are not limited to those selected from the groupconsisting of benzyl alcohol, sorbic acid, parabens, imidurea andcombinations thereof. A particularly preferred preservative is acombination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5%sorbic acid.

The composition preferably includes an antioxidant and a chelatingagent, which inhibit the degradation of the compound. Preferredantioxidants for some compounds are BHT, BHA, alpha-tocopherol andascorbic acid in the preferred range of about 0.01% to 0.3% and morepreferably BHT in the range of 0.03% to 0.2% by weight by total weightof the composition. Preferably, the chelating agent is present in anamount ranging from 0.01% to 0.5% by weight by total weight of thecomposition. Particularly preferred chelating agents include edetatesalts (e.g. disodium edetate) and citric acid in the weight range ofabout 0.01% to 0.20% and more preferably in the range of 0.02% to 0.10%by weight by total weight of the composition. The chelating agent isuseful for chelating metal ions in the composition, which may bedetrimental to the shelf life of the formulation. While BHT and disodiumedetate are the particularly preferred antioxidant and chelating agentrespectively for some compounds, other suitable and equivalentantioxidants and chelating agents may be substituted therefore as wouldbe known to those skilled in the art.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water, and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives (e.g., sodium carboxymethylcellulose,hydroxypropylmethylcellulose, methylcellulose). Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin, and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. As used herein, an “oily” liquidis one that comprises a carbon-containing liquid molecule and whichexhibits a less polar character than water. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water, and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Administration/Dosing

The regimen of administration may affect what constitutes an effectiveamount. For example, several divided dosages, as well as staggereddosages may he administered daily or sequentially, or the dose may becontinuously infused, or may be a bolus injection. Further, the dosagesof the therapeutic formulations may be proportionally increased ordecreased as indicated by the exigencies of the therapeutic orprophylactic situation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat a disease or disorder in the patient. An effective amount ofthe therapeutic compound necessary to achieve a therapeutic effect mayvary according to factors such as the activity of the particularcompound employed; the time of administration; the rate of excretion ofthe compound; the duration of the treatment; other drugs, compounds ormaterials used in combination with the compound; the state of thedisease or disorder, age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell-known in the medical arts. Dosage regimens may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation. Anon-limiting example of an effective dose range for a therapeuticcompound of the invention is from about 0.01 and 50 mg/kg of bodyweight/per day. One of ordinary skill in the art would be able to studythe relevant factors and make the determination regarding the effectiveamount of the therapeutic compound without undue experimentation.

The compound can be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on. The frequency of the dose is readilyapparent to the skilled artisan and depends upon any number of factors,such as, but not limited to, the type and severity of the disease beingtreated, and the type and age of the animal.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

A medical doctor, physician or veterinarian, having ordinary skill inthe art may readily determine and prescribe the effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of a disease or disorder in a patient.

In certain embodiments, the compositions of the invention areadministered to the patient in dosages that range from one to five timesper day or more. In other embodiments, the compositions of the inventionare administered to the patient in range of dosages that include, butare not limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It is readily apparent to oneskilled in the art that the frequency of administration of the variouscombination compositions of the invention varies from subject to subjectdepending on many factors including, but not limited to, age, disease ordisorder to be treated, gender, overall health, and other factors. Thus,the invention should not he construed to he limited to any particulardosage regime and the precise dosage and composition to be administeredto any patient will he determined by the attending physical taking allother factors about the patient into account.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 7,500 mg, about 20 μg to about 7,000 mg, about40 μg, to about 6,500 mg, about 80 μg to about 6,000 mg, about 100 μg toabout 5,500 mg, about 200 μg to about 5,000 mg, about 400 μg to about4,000 mg, about 800 μg to about 3,000 mg, about 1 mg to about 2,500 mg,about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mgto about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about150 mg, and any and all whole or partial increments therebetween.

In some embodiments, the dose of a compound of the invention is fromabout 0.5 μg and about 5,000 mg. In some embodiments, a dose of acompound of the invention used in compositions described herein is lessthan about 5,000 mg, or less than about 4,000 mg, or less than about3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, orless than about 800 mg, or less than about 600 mg, or less than about500 mg, or less than about 200 mg, or less than about 50 mg. Similarly,one embodiments, a dose of a second compound as described herein is lessthan about 1,000 mg, or less than about 800 mg, or less than about 600mg, or less than about 500 mg, or less than about 400 mg, or less thanabout 300 mg, or less than about 200 mg, or less than about 100 mg, orless than about 50 mg, or less than about 40 mg, or less than about 30mg, or less than about 25 mg, or less than about 20 mg, or less thanabout 15 mg, or less than about 10 mg, or less than about 5 mg, or lessthan about 2 mg, or less than about I mg, or less than about 0.5 mg, andany and all whole or partial increments thereof.

In certain embodiments, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof a disease or disorder in a patient.

The term “container” includes any receptacle for holding thepharmaceutical composition. For example, in certain embodiments, thecontainer is the packaging that contains the pharmaceutical composition.In other embodiments, the container is not the packaging that containsthe pharmaceutical composition, i.e., the container is a receptacle,such as a box or vial that contains the packaged pharmaceuticalcomposition or unpackaged pharmaceutical composition and theinstructions for use of the pharmaceutical composition. Moreover,packaging techniques are well known in the art. It should be understoodthat the instructions for use of the pharmaceutical composition may hecontained on the packaging containing the pharmaceutical composition,and as such the instructions form an increased functional relationshipto the packaged product. However, it should be understood that theinstructions may contain information pertaining to the compound'sability to perform its intended function, e.g., treating, preventing, orreducing a disease or disorder in a patient.

Routes of Administration

Routes of administration of any of the compositions of the inventioninclude inhalational, oral, nasal, rectal, parenteral, sublingual,transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal,(trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal,and (trans)rectal), intravesical, intrapulmonary, intraduodenal,intragastrical, intrathecal, subcutaneous, intramuscular, intradermal,intra-arterial, intravenous, intrabronchial, inhalation, and topicaladministration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Otherformulations suitable for oral administration include, but are notlimited to, a powdered or granular formulation, an aqueous or oilysuspension, an aqueous or oily solution, a paste, a gel, toothpaste, amouthwash, a coating, an oral rinse, or an emulsion. The compositionsintended for oral use may be prepared according to any method known inthe art and such compositions may contain one or more agents selectedfrom the group consisting of inert, non-toxic pharmaceuticallyexcipients that are suitable for the manufacture of tablets. Suchexcipients include, for example an inert diluent such as lactose;granulating and disintegrating agents such as cornstarch; binding agentssuch as starch; and lubricating agents such as magnesium stearate.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmoticallycontrolled release tablets. Tablets may further comprise a sweeteningagent, a flavoring agent, a coloring agent, a preservative, or somecombination of these in order to provide for pharmaceutically elegantand palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

For oral administration, the compounds of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents; fillers;lubricants; disintegrates; or wetting agents. If desired, the tabletsmay be coated using suitable methods and coating materials such asOPADRY™ film coating systems available from Colorcon, West Point, Pa.(e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, AqueousEnteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400).

Liquid preparation for oral administration may be in the form ofsolutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propylpara-hydroxy benzoates or sorbic acid). Liquid formulations of apharmaceutical composition of the invention which are suitable for oraladministration may be prepared, packaged, and sold either in liquid formor in the form of a dry product intended for reconstitution with wateror another suitable vehicle prior to use.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface-active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or inciting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) will melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds usefulwithin the methods of the invention, and a further layer providing forthe immediate release of one or more compounds useful within the methodsof the invention. Using a wax/pH-sensitive polymer mix, a gastricinsoluble composition may be obtained in which the active ingredient isentrapped, ensuring its delayed release.

Parenteral Administration

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intravenous, intraperitoneal, intramuscular, intrasternal injection, andkidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may he formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butanediol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer system. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Additional Administration Forms

Additional dosage forms of this invention include dosage forces asdescribed in U.S. Pat. Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389,5,582,837, and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041, WO03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO98/11879, WO 97/47285, WO 93/18755, and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.In some cases, the dosage forms to be used can be provided as slow orcontrolled-release of one or more active ingredients therein using, forexample, hydropropylmethyl cellulose, other polymer matrices, gels,permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, or microspheres or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the pharmaceutical compositions of the invention. Thus, single unitdosage forms suitable for oral administration, such as tablets,capsules, gelcaps, and caplets, which are adapted for controlled-releaseare encompassed by the present invention.

Most controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood level of the drug, andthus can affect the occurrence of side effects.

Most controlled-release formulations are designed to initially releasean amount of drug that promptly produces the desired therapeutic effect,and gradually and continually release of other amounts of drug tomaintain this level of therapeutic effect over an extended period oftime. In order to maintain this constant level of drug in the body, thedrug must be released from the dosage form at a rate that will replacethe amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by variousinducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. The term “controlled-releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, or microspheresor a combination thereof that facilitates the controlled-release of theactive ingredient.

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release which is longer that the same amount of agent administeredin bolus form. For sustained release, the compounds may be formulatedwith a suitable polymer or hydrophobic material which providessustained, release properties to the compounds. As such, the compoundsfor use the method of the invention may be administered in the form ofmicroparticles, for example, by injection or in the form of wafers ordiscs by implantation. In a preferred embodiment of the invention, thecompounds of the invention are administered to a patient, alone or incombination with another pharmaceutical agent, using a sustained releaseformulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours. The term pulsatile release is used herein in itsconventional sense to refer to a drug formulation that provides releaseof the drug in such a way as to produce pulsed plasma profiles of thedrug after drug administration. The term immediate release is used inits conventional sense to refer to a drug formulation that provides forrelease of the drug immediately after drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Methods and Materials: Sequences:

Extracellular domain of ENPP3 (SEQ ID NO: 1) EKQGSCRKKC FDASFRGLENCRCDVAC KDRGDCCWDF EDTCVESTRI WMCNKFRCGE TRLEASLCSC SDDCLQRKDCCADYKSVCQG ETSWLEENCD TAQQSQCPEG FDLPPVILFS MDGFRAEYLY TWDTLMPNINKLKTCGIHSK YMRAMYPTKT FPNHYTIVTG LYPESHGIID NNMYDVNLNK NFSLSSKEQNNPAWWHGQPM NLTAMYQGLK AATYFWPGSE VAINGSFPSI YMPYNGSVPF EERISTLLKWLDLPKAERPR FYTMYFEEPD SSGHAGGPVS ARVIKALQVV DHAFGMLMEG LKQRNLHNCVNIILLADHGM DQTYCNKMEY MTDYFPRINF FYMYEGPAPR IRAHNIPHDF FSFNSEEIVRNLSCRKPDQH FKPYLTPDLP KRLHYAKNVR IDKVHLFVDQ QWLAVRSKSN TNCGGGNHGYNNEFRSMEAI FLAHGPSFKE KTEVEPFENI EVYNLMCDLL RIQPAPNNGT HGSLNHLLKVPFYEPSHAEE VSKFSVCGFA NPLPTESLDC FCPHLQNSTQ LEQVNQMLNL TQEEITATVKVNLPFGRPRV LQKNVDHCLL YHREYVSGFG KAMRMPMWSS YTVPQLGDTS PLPPTVPDCLRADVRVPPSE SQKCSFYLAD KNITHGFLYP PASNRTSDSQ YDALITSNLV PMYEEFRKMWDYFHSVLLIK HATERNGVNV VSGPIFDYNY DGHFDAPDEI TKHLANTDVP IPTHYFVVLTSCKNKSHTPE NCPGWLDVLP FIIPHRPTNV ESCPEGKPEA LWVEERFTAH IARVRDVELLTGLDFYQDKV QPVSEILQLK TYLPTFETTI Signal sequence ENPP7 (SEQ ID NO: 2)MRGPAVLLTV ALATLLAPGA Signal sequence ENPP7 (SEQ ID NO: 3)MRGPAVLLTV ALATLLAPGA GA Signal Sequence ENPP5 (SEQ ID NO: 4)MTSKFLLVSF ILAALSLSTT FS Signal Sequence ENPP1-2-1  (SEQ ID NO: 5)M E R D G C A G G G S R G G E G G R A P R E GP A G N G R D R G R S H A A E A P G D P Q A AA S L L A P M D V G E E P L E K A A R A R T AK D P N T Y K I I S L F T F A V G V N I C L G F T A(singly underlined)-(doubly underlined): Swapped residues with NPP2 residues 1-27to give cleavage at the singly underlined-doubly underlined transitionSEQ ID NO: 6(DSS)_(n), wherein n is an integer ranging between 1 and 20.SEQ ID NO: 7(ESS)_(n), wherein n is an integer ranging between 1 and 20.SEQ ID NO: 8(RQQ)_(n), wherein n is an integer ranging between 1 and 20.SEQ ID NO: 9 (KR)_(n), wherein n is an integer ranging between 1 and 20.SEQ ID NO: 10 R_(n), wherein n is an integer ranging between 1 and 20.SEQ ID NO: 11(KR)_(n), wherein n is an integer ranging between 1 and 20.SEQ ID NO: 12 DSSSEEKFLRRIGRFG SEQ ID NO: 13 EEEEEEEPRGDT SEQ ID NO: 14APWHLSSQYSRT SEQ ID NO: 15 STLPIPHEFSRE SEQ ID NO: 16 VTKHLNQISQSYSEQ ID NO: 17 E_(n), wherein n is an integer ranging between 1 and 20.SEQ ID NO: 18 D_(n), wherein n is an integer ranging between 1 and 20.ENPP121-NPP3-Fc sequence  (SEQ ID NO: 19)MERDGCAGGG SRGGEGGRAP REGPAGNGRD RGRSHAAEAP GDPQAAASLL APMDVGEEPLEKAARARTAK DPNTYKIIS L FTFAVGVNIC LGFTAKQGSC RKKCFDASFR GLENCRCDVACKDRGDCCWD FEDTCVESTR IWMCNKFRCG ERLEASLCSC SDDCLQRKDC CADYKSVCQGETSWLEENCD TAQQSQCPEG FDLPPVILFS MDGFRAEYLY TWDTLMPNIN KLKTCGIHSKYMRAMYPTKT FPNHYTIVTG LYPESHGIID NNMYDVNLNK NFSLSSKEQN NPAWWHGQPMWLTAMYQGLK AATYFWPGSE VAINGSFPSI YMPYNGSVPF EERISTLLKW LDLPKAERPRFYTMYEEEPD SSGHAGGPVS ARVIKALQVV DHAFGMLMEG LKQRNLHNCV NIILLADHGMDQTYCNKMEY MTDYFPRINF FYMYEGPAPR IRAHNIPHDF FSFNSEEIVR NLSCRKPDQHFKPYLTPDLP KRLHYAKNVR IDKVHLFVDQ QWLAVRSKSN TNCGGGNHGY NNEFRSMEAIFLAHGPSFKE KTEVEPFENI EVYNLMCDLL RIQPAPNNGT HGSLNHLLKV PFYEPSHAEEVSKFSVCGFA NPLPTESLDC FCPHLQNSTQ LEQVNQMLNL TQEEITATVK VNLPFGRPRVLQKNVDHCLL YHREYVSGFG KAMRMPMWSS YTVPQLGDTS PLPPTVEDCL RADVRVPPSESQKCSFYLAD KNITHGFLYP PASNRTSDSQ YDALITSNLV PMYEEFRKMW DYFHSVLLIKHATERNGVNV VSGPIFDYNY DGHFDAPDEI TKHLANTDVP IPTHYFVVLT SCKNKSHTPENCPGWLDVLP FIIPHRPTNV ESCPEGKPEA LWVEERFTAH IARVRDVELL TGLDFYQDKVQPVSEILQLK TYLPTFETTI DKTHTCPPCP APELLGGPSV FLFTPKTKDT LMISRTPEVTCVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYECKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKSLSLSPGK

Bold residues=amino acid sequence from NPP1; Single underlinedresidues=signal peptide sequence from NPP2; Double underlinedresidues=amino acid sequence of IgG Fe domain. In certain embodiments,the IgG Fe domain is selected from any of the subclasses IgG1, IgG2,IgG3 and IgG4. In other embodiments, instead of Fc domain, albumindomain is used.

In certain embodiments, the NPP3 C-terminus and the Fc/albumin domainare connected by a linker. In other embodiments, the linker comprises atleast two amino acids. In yet other embodiments, the linker comprises2-40 amino acids, 2-30 amino acids, 2-20 amino acids, 2-18 amino acids,2-16 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10 amino acids,2-8 amino acids, 2-6 amino acids, 2-4 amino acids, or 2 amino acids. Inyet other embodiments, the flexible linker comprises a polyethyleneglycol chain and/or a hydrocarbon chain (such as an alkylene chain).

IgG Fc sequence (SEQ ID NO: 20)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ENPP7-NPP3-Fc sequence (SEQ ID NO: 21)MRGPAVLLTV ALATLLAPGA KQGSC RKKCFDASFR GLENCRCDVACKDRGDCCWD FEDTCVESTR IWMCNKFRCG ERLEASLCSC SDDCLQRKDC CADYKSVCQGETSWLEENCD TAQQSQCPEG FDLPPVILFS MDGFRAEYLY TWDTLMPNIN KLKTCGIHSKYMRAMYPTKT FPNHYTIVTG LYPESHGIID NNMYDVNLNK NFSLSSKEQN NPAWWHGQPMWLTAMYQGLK AATYFWPGSE VAINGSFPSI YMPYNGSVPF EERISTLLKW LDLPKAERPRFYTMYFEEPD SSGHAGGPVS ARVIKALQVV DHAFGMLMEG LKQRNLHNCV NIILLADHGMDQTYCNKMEY MTDYFPRINF FYMYEGPAPR IRAHNIPHDF FSFNSEEIVR NLSCRKPDQHFKPYLTPDLP KRLHYAKNVR IDKVHLFVDQ QWLAVRSKSN TNCGGGNHGY NNEFRSMEAIFLAHGPSFKE KTEVEPFENI EVYNLMCDLL RIQPAPNNGT HGSLNHLLKV PFYEPSHAEEVSKFSVCGFA NPLPTESLDC FCPHLQNSTQ LEQVNQMLNL TQEEITATVK VNLPFGRPRVLQKNVDHCLL YHREYVSGFG KAMRMPMWSS YTVPQLGDTS PLPPTVPDCL RADVRVPPSESQKCSFYLAD KNITHGFLYP PASNRTSDSQ YDALITSNLV PMYEEFRKMW DYFHSVLLIKHATEPNGVNV VSGPIFDYNY DGHFDAPDEI TKHLANTDVP IPTHYFVVLT SCKNKSHTPENCPGWLDVLP FIIPHRPTNV ESCPEGKPEA LWVEERFTAH IARVRDVELL TGLDFYQDKVQPVSEILQLK TYLPTFETTI DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVTCVVVDVSHED PEVKFNWYVD GVEVHSNKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKSLSLSPGK

Single underlined residues=signal peptide sequence from NPP7; Doubleunderlined residues=amino acid sequence of IgG Fc domain. In certainembodiments, the IgG Fc domain is selected from any of the subclassesIgG1, IgG2, IgG3 and IgG4. In other embodiments, instead of Fc domain,albumin domain is used.

In certain embodiments, the NPP3 C-terminus and the Fc/albumin domainare connected by a linker. In other embodiments, the linker comprises atleast two amino acids. In yet other embodiments, the linker comprises2-40 amino acids, 2-30 amino acids, 2-20 amino acids, 2-18 amino acids,2-16 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10 amino acids,2-8 amino acids, 2-6 amino acids, 2-4 amino acids, or 2 amino acids. Inyet other embodiments, the flexible linker comprises a polyethyleneglycol chain and/or a hydrocarbon chain (such as an alkylene chain).

ENPP5-NPP3-Fc sequence (SEQ ID NO: 22)MTSKFLLVSF ILAALSLSTT FSKQGSC RKKCFDASFR GLENCRCDVACKDRGDCCWD FEDTCVESTR IWMCNKFRCG ERLEASLCSC SDDCLQRKDC CADYKSVCQGETSWLEENCD TAQQSQCPEG FDLPPVILFS MDGFRAEYLY TWDTLMPNIN KLKTCGIHSKYMRAMYPTKT FPNHYTIVTG LYPESHGIID NNMYDVNLNK NTSLSSKEQN NPAWWHGQPMWLTAMYQGLK AATYFWPGSE VAINGSFPSI YMPYNGSVPF EERISTLLKW LDLPKAERPRFYTMYFEEPD SSGHAGGPVS ARVIKALQVV DHAFGMLMEG LKQRNLHNCV NIILLADHGMDQTYCNKMEY MTDYFPRINF FYMYEGPAPR IRAHNIPHDF FSFNSEEIVR NLSCRKPDQHFKPYLTPDLP KRLHYAKNVR IDKVHLFVDQ QWLAVRSKSN TNCGGGNHGY NNEFRSMEAIFLAHGPSFKE KTEVEPFFNI EVYNLMCDLL RIQPAPNNGT HGSLNHLLKV PFYEPSHAEEVSKFSVCGFA NPLPTESLDC FCPHLQNSTQ LEQVNQMLNL TQEEITATVK VNLPFGRPRVLQKNVDHCLL YHREYVSGFG KAMRMPMWSS YTVPQLGDTS PLPPTVPDCL RADVRVPPSESQKCSFYLAD KNITHGFLYP PASNRTSDSQ YDALITSNLV PMYEEFRKMW DYFHSVLLIKHATERNGVNV VSGPIFDYNY DGHFDAPDEI TKHLANTDVP IPTHYFVVLT SCKNKSHTPENCPGWLDVLP FIIPHRPTNV ESCPEGKPEA LWVEERFTAH IARVRDVELL TGLDFYQDKVQPVSEILQLK TYLPTFETTI DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVTCVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKSLSLSPGK

Single underlined residues=signal peptide sequence from NPP5; Doubleunderlined residues=amino acid sequence of IgG Fc domain. In certainembodiments, the IgG Fc domain is selected from any of the subclassesIgG1, IgG2, IgG3 and IgG4. In other embodiments, instead of Fe domain,albumin domain is used.

In certain embodiments, the NPP3 C-terminus and the Fe/albumin domainare connected by a linker. In other embodiments, the linker comprises atleast two amino acids. In yet other embodiments, the linker comprises2-40 amino acids, 2-30 amino acids, 2-20 amino acids. 2-18 amino acids,2-16 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10 amino acids,2-8 amino acids, 2-6 amino acids, 2-4 amino acids, or 2 amino acids. Inyet other embodiments, the flexible linker comprises a polyethyleneglycol chain and/or a hydrocarbon chain (such as an alkylene chain).

Albumin sequence (SEQ ID NO: 23)GGGGSGGGGSGGGGSMKWVTFLLLLFVSGSAFSRGVFRREAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA ENPP121-NPP3-Albumin sequence (SEQ ID NO: 24)MERDGCAGGG SRGGEGGRAP REGPAGNGRD RGRSHAAEAP GDPQAAASLL APMDVGEEPLEKAARARTAK DPNTYKIIS L FTFAVGVNIC LGFTAKQGSC RKKCFDASFR GLENCRCDVACKDRGDCCWD FEDTCVESTR IWMCNKFRCG ERLEASLCSC SDDCLQRKDC CADYKSVCQGETSWLEENCD TAQQSQCPEG FDLPPVILFS MDGFRAEYLY TWDTLMPNIN KLKTCGIHSKYMRAMYPTKT FPNHYTIVTG LYPESHGIID NNMYDVNLNK NFSLSSKEQN NPAWWHGQPMWLTAMYQGLK AATYFWPGSE VAINGSFPSI YMPYNGSVPF EERISTLLKW LDLPKAERPRFYTMYFEEPD SSGHAGGPVS ARVIKALQVV DHAFGMLMEG LKQRNLHNCV NIILLADHGMDQTYCNKMEY MTDYFPRINF FYMYEGPAPR IRAHNIPHDF FSFNSEEIVR NLSCRKPDQHFKPYLTPDLP KRLHYAKNVR IDKVHLFVDQ QWLAVRSKSN TNCGGGNHGY NNEFRSMEAIFLAHGPSFKE KTEVEPFENI EVYNLMCDLL RIQPAPNNGT HGSLNHLLKV PFYEPSHAEEVSKFSVCGFA NPLPTESLDC FCPHLQNSTQ LEQVNQMLNL TQEEITATVK VNLPFGRPRVLQKNVDHCLL YHREYVSGFG KAMRMPMWSS YTVPQLGDTS PLPPTVPDCL RADVRVPPSESQKCSFYLAD KNITHGFLYP PASNRTSDSQ YDALITSNLV PMYEEFRKMW DYFHSVLLIKHATERNGVNV VSGPIFDYNY DGHFDAPDEI TKHLANTDVP IPTHYFVVLT SCKNKSHTPENCPGWLDVLP FIIPHRPTNV ESCPEGKPEA LWVEERFTAH IARVRDVELL TGLDFYQDKVQPVSEILQLK TYLPTFETTIGGGSGGGGSG GGGSMKWVTF LLLLFVSGSA FSRGVFRREA HKSEIAHRYN DLGEQHFKGLVLIAFSQYLQ KCSYDEHAKL VQEVTDFAKT CVADESAANC DKSLHTLFGD KLCAIPNLRENYGELADCCT KQEPERNECF LQHKDDNPSL PPFERPEAEA MCTSFKENPT TFMGHYLHEVARRHPYFYAP ELLYYAEQYN EILTQCCAEA DKESCLTPKL DGVKEKALVS SVRQRMKCSSMQKFGERAFK AWAVARLSQT FPNADFAEIT KLATDLTKVN KECCHGDLLE CADDRAELAKYMCENQATIS SKLQTCCDKP LLKKAHCLSE VEHDTMPADL PAIAADFVED QEVCKNYAEAKDVFLGTFLY EYSRRHPDYS VSLLLRLAKK YEATLEKCCA EANPPACYGT VLAEFQPLVEEPKNLVKTNC DLYEKLGEYG FQNAILVRYT QKAPQVSTPT LVEAARNLGR VGTKCCTLPEDQRLPCVEDY LSAILNRVCL LHEKTPVSEH VTKCCSGSLV ERRPCFSALT VDETYVPKEFKAETFTEHSD ICTLPEKEKQ IKKQTALAEL VKHKPKATAE QLKTVMDDFA QFLDTCCKAADKDTCFSTEG PNLVTRCKDA LA

Bold residues=amino acid sequence from NPP1; Single underlinedresidues=signal peptide sequence from NPP2; Double underlinedresidues=amino acid sequence of spacer sequence and albumin domain.

In certain embodiments, the NPP3 C-terminus and the albumin domain areconnected by a linker. In other embodiments, the linker comprises atleast two amino acids. In yet other embodiments, the linker comprises2-40 amino acids, 2-30 amino acids, 2-20 amino acids, 2-18 amino acids,2-16 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10 amino acids,2-8 amino acids, 2-6 amino acids, 2-4 amino acids, or 2 amino acids. Inyet other embodiments, the flexible linker comprises a polyethyleneglycol chain and/or a hydrocarbon chain (such as an alkylene chain).

ENPP7-NPP3-Albumin sequence (SEQ ID NO: 25)MRGPAVLLTV ALATLLAPGA KQGSC RKKCFDASFR GLENCRCDVACKDRGDCCWD FEDTCVESTR IWMCNKFRCG ERLEASLCSC SDDCLQRKDC CADYKSVCQGETSWLEENCD TAQQSQCPEG FDLPPVILFS MDGFRAEYLY TWDTLMPNIN KLKTCGIHSKYMRAMYPTKT FPNHYTIVTG LYPESHGIID NNMYDVNLNK NFSLSSKEQN NPAWWHGQPMWLTAMYQGLK AATYFWPGSE VAINGSFPSI YMPYNGSVPF EERISTLLKW LDLPKAERPRFYTMYFEEPD SSGHAGGPVS ARVIKALQVV DHAFGMLMEG LKQRNLHNCV NIILLADHGMDQTYCNKMEY MTDYFPRINF FYMYEGPAPR IRAHNIPHDF FSFNSEEIVR NLSCRKPDQHFKPYLTPDLP KRLHYAKNVR IDKVHLFVDQ QWLAVRSKSN TNCGGGNHGY NNEFRSMEAIFLAHGPSFKE KTEVEPFENI EVYNLMCDLL RIQPAPNNGT HGSLNHLLKV PFYEPSHAEEVSKFSVCGFA NPLPTESLDC FCPHLQNSTQ LEQVNQMLNL TQEEITATVK VNLPFGRPRVLQKNVDHCLL YHREYVSGFG KAMRMPMWSS YTVPQLGDTS PLPPTVPDCL RADVRVPPSESQKCSFYLAD KNITHGFLYP PASNRTSTSQ YDALITSNLV PMYEEFRKMW DYFHSVLLIKHATERNGVNV VSGPIFDYNY DGHFDAPDEI TKHLANTDVP IPTHYFVVLT SCKNKSHTPENCPGWLDVLP FIIPHRPTNV ESCPEGKPEA LWVEERFTAH LARVRDVELL TGLDFYQDKVQPVSEILQLK TYLPTFETTIGGGSGGGGSG GGGSMKWVTF LLLLFVSGSA FSRGVFRREA HKSEIAHRYN DLGEQHFKGLVLIAFSQYLQ KCSYDEHAKL VQEVTDFAKT CVADESAANC DKSLHTLFGD KLCAIPNLRENYGELADCCT KQEPERNECF LQHKDDNPSL PPFERPEAEA MCTSFKENPT TFMGHYLHEVARRHPYFYAP ELLYYAEQYN EILTQCCAEA DKESCLTPKL DGVKEKALVS SVRQRMKCSSMQKFGERAFK AWAVARLSQT FPNADFAEIT KLATDLTKVN KECCHGDLLE CADDRAELAKYMCENQATIS SKLQTCCDKP LLKKAHCLSE VEHDTMPADL PAIAADFVED QEVCKNYAEAKDVFLGTFLY EYSRRHPDYS VSLLLRLAKK YEATLEKCCA EANPPACYGT VLAEFQPLVEEPKNLVKTNC DLYEKLGEYG FQNAILVRYT QKAPQVSTPT LVEAARNLGR VGTKCCTLPEDQRLPCVEDY LSAILNRVCL LHEKTPVSEH VTKGCSGSLV ERRPCFSALT VDETYVPKEFKAETFTFHSD ICTLPEKEKQ IKKQTALAEL VKHKPKATAE QLKTVMDDFA QFLDTCCKAADKDTCFSTEG PNLVTRCKDA LA

Single underlined residues=signal peptide sequence from NPP7; Doubleunderlined residues=amino acid sequence of spacer sequence and albumindomain.

In certain embodiments, the NPP3 C-terminus and the albumin domain areconnected by a linker. In other embodiments, the linker comprises atleast two amino acids. In yet other embodiments, the linker comprises2-40 amino acids, 2-30 amino acids, 2-20 amino acids, 2-18 amino acids,2-16 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10 amino acids,2-8 amino acids, 2-6 amino acids, 2-4 amino acids, or 2 amino acids. Inyet other embodiments, the flexible linker comprises a polyethyleneglycol chain and/or a hydrocarbon chain (such as an alkylene chain).

ENPP5-NPP3-albumin sequence (SEQ ID NO: 26)MTSKFLLVSF ILAALSLSTT FSKQGSC RKKCFDASFR GLENCRCDVACKDRGDCCWD FEDTCVESTR IWMCNKFRCG ERLEASLCSC SDDCLQRKDC CADYKSVCQGETSWLEENCD TAQQSQCPEG FDLPPVILFS MDGFRAEYLY TWDTLMPNIN KLKTCGIHSKYMRAMYPTKT FPNHYTIVTG LYPESHGIID NNMYDVNLNK NFSISSKEQN NPAWWHGQPMWLTAMYQGLK AATYFWPGSE VAINGSFPSI YMPYNGSVPF EERISTLLKW LDLPKAERPRFYTMYFEEPD SSGHAGGPVS ARVIKALQVV DHAFGMLMEG LKQRNLHNCV NIILLADHGMDQTYCNKMEY MTDYFPRINF FYMYEGPAPR IRAHNIPHDF FSFNSEEIVR NLSCRKPDQHFKPYLTPDLP KRLHYAKNVR IDKVHLFVDQ QWLAVRSKSN TNCGGGNHGY NNEFRSMEAIFLAHGPSFKE KTEVEPFENI EVYNLMCDLL RIQPAPNNGT HGSLNHLLKV PFYEPSHAEEVSKFSVCGFA NPLPTESLDC FCPHLQNSTQ LEQVNQMLNL TQEEITATVK VNLPFGRPRVLQKNVDHCLL YHREYVSGFG KAMRMPMWSS YTVPQLGDTS PLPPTVPDCL RADVRVPPSESQKCSFYLAD KNITHGFLYP PASNRTSDSQ YDALITSNLV PMYEEFRKMW DYFHSVLLIKHATERNGVNV VSGPIFDYNY DGHFDAPDEI TKHLANTDVP IPTHYFVVLT SCKNKSHTPENCPGWLDVLP FIIPHRPTNV ESCPEGKPEA LWVEERFTAH IARVRDVELL TGLDFYQDKVQPVSEILQLK TYLPTFETTIGGGSGGGGSG GGGSMKWVTF LLLLFVSGSA FSRGVFRREA HKSEIAHRYN DLGEQHFKGLVLIAFSQYLQ KCSYDEHAKL VQEVTDFAKT CVADESAANC DKSLHTLFGD KLCAIPNLRENYGELADCCT KQEPERNECF LQHKDDNPSL PPFERPEAEA MCTSFKENPT TFMGHYLHEVARRHPYFYAP ELLYYAEQYN EILTQCCAEA DKESCLTPKL DGVKEKALVS SVRQRMKCSSMQKFGERAFK AWAVARLSQT FPNADFAEIT KLATDLTKVN KECCHGDLLE CADDRAELAKYMCENQATIS SKLQTCCDKP LLKKAHCLSE VEHDTMPADL PAIAADFVED QEVCKNYAEAKDVFLGTFLY EYSRRHPDYS VSLLLRLAKK YEATLEKCCA EANPPACYGT VLAEFQPLVEEPKNLVKTNC DLYEKLGEYG FQNAILVRYT QKAPQVSTPT LVEAARNLGR VGTKCCTLPEDQRLPCVEDY LSAILNRVCL LHEKTPVSEH VTKGCSGSLV ERRPCFSALT VDETYVPKEFKAETFTFHSD ICTLPEKEKQ IKKQTALAEL VKHKPKATAE QLKTVMDDFA QFLDTCCKAADKDTCFSTEG PNLVTRCKDA LA

Single underlined residues=signal peptide sequence from NPP5; Doubleunderlined residues=amino acid sequence of spacer sequence and albumindomain. In certain embodiments, the NPP3 C-terminus and the albumindomain are connected by a linker. In other embodiments, the linkercomprises at least two amino acids. In yet other embodiments, the linkercomprises 2-40 amino acids, 2-30 amino acids, 2-20 amino acids, 2-18amino acids, 2-16 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10amino acids, 2-8 amino acids, 2-6 amino acids, 2-4 amino acids, or 2amino acids. In yet other embodiments, the flexible linker comprises apolyethylene glycol chain and/or a hydrocarbon chain (such as analkylene chain).

Nucleotide sequence of NPP121-NPP3-Fc (SEQ ID NO: 27)ATGGAAAGGGACGGATGCGCCGGTGGTGGATCTCGCGGAGGCGAAGGTGGAAGGGCCCCTAGGGAAGGACCTGCCGGAAACGGAAGGGACAGGGGACGCTCTCACGCCGCTGAAGCTCCAGGCGACCCTCAGGCCGCTGCCTCTCTGCTGGCTCCTATGGACGTCGGAGAAGAACCCCTGGAAAAGGCCGCCAGGGCCAGGACTGCCAAGGACCCCAACACCTACAAGATCATCTCCCTCTTCACTTTCGCCGTCGGAGTCAACATCTGCCTGGGATTCACCGCCGAAAAGCAAGGCAGCTGCAGGAAGAAGTGCTTTGATGCATCATTTAGAGGACTGGAGAACTGCCGGTGTGATGTGGCATGTAAAGACCGAGGTGATTGCTGCTGGGATTTTGAAGACACCTGTGTGGAATCAACTCGAATATGGATGTGCAATAAATTTCGTTGTGGAGAGACCAGATTAGAGGCCAGCCTTTGCTCTTGTTCAGATGACTGTTTGCAGAGGAAAGATTGCTGTGCTGACTATAAGAGTGTTTGCCAAGGAGAAACCTCATGGCTGGAAGAAAACTGTGACACAGCCCAGCAGTCTCAGTGCCCAGAAGGGTTTGACCTGCCACCAGTTATCTTGTTTTCTATGGATGGATTTAGAGCTGAATATTTATACACATGGGATACTTTAATGCCAAATATCAATAAACTGAAAACATGTGGAATTCATTCAAAATACATGAGAGCTATGTATCCTACCAAAACCTTCCCAAATCATTACACCATTGTCACGGGCTTGTATCCAGAGTCACATGGCATCATTGACAATAAAATGTATGATGTAAATCTCAACAAGAATTTTTCACTTTCTTCAAAGGAACAAAATAATCCAGCCTGGTGGCATGGGCAACCAATGTGGCTGACAGCAATGTATCAAGGTTTAAAAGCCGCTACCTACTTTTGGCCCGGATCAGAAGTGGCTATAAATGGCTCCTTTCCTTCCATATACATGCCTTACAACGGAAGTGTCCCATTTGAAGAGAGGATTTCTACACTGTTAAAATGGCTGGACCTGCCCAAAGCTGAAAGACCCAGGTTTTATACCATGTATTTTGAAGAACCTGATTCCTCTGGACATGCAGGTGGACCAGTCAGTGCCAGAGTAATTAAAGCCTTACAGGTAGTAGATCATGCTTTTGGGATGTTGATGGAAGGCCTGAAGCAGCGGAATTTGCACAACTGTGTCAATATCATCCTTCTGGCTGACCATGGAATGGACCAGACTTATTGTAACAAGATGGAATACATGACTGATTATTTTCCCAGAATAAACTTCTTCTACATGTACGAAGGGCCTGCCCCCCGCATCCGAGCTCATAATATACCTCATGACTTTTTTAGTTTTAATTCTGAGGAAATTGTTAGAAACCTCAGTTGCCGAAAACCTGATCAGCATTTCAAGCCCTATTTGACTCCTGATTTGCCAAAGCGACTGCACTATGCCAAGAACGTCAGAATCGACAAAGTTCATCTCTTTGTGGATCAACAGTGGCTGGCTGTTAGGAGTAAATCAAATACAAATTGTGGAGGAGGCAACCATGGTTATAACAATGAGTTTAGGAGCATGGAGGCTATCTTTCTGGCACATGGACCCAGTTTTAAAGAGAAGACTGAAGTTGAACCATTTGAAAATATTGAAGTCTATAACCTAATGTGTGATCTTCTACGCATTCAACCAGCACCAAACAATGGAACCCATGGTAGTTTAAACCATCTTCTGAAGGTGCCTTTTTATGAGCCATCCCATGCAGAGGAGGTGTCAAAGTTTTCTGTTTGTGGCTTTGCTAATCCATTGCCCACAGAGTCTCTTGACTGTTTCTGCCCTCACCTACAAAATAGTACTCAGCTGGAACAAGTGAATCAGATGCTAAATCTGACCCAAGAAGAAATAACAGGAACAGTGAAAGTAAATTTGCCATTTGGGAGGCCTAGGGTACTGCAGAAGAACGTGGACCACTGTCTCCTTTACCACAGGGAATATGTCAGTGGATTTGGAAAAGCTATGAGGATGCCCATGTGGAGTTCATACACAGTCCCCCAGTTGGGAGACACATCGCCTCTGCCTCCCACTGTCCCAGACTGTCTGCGGGCTGATGTCAGGGTTCCTCCTTCTGAGAGCCAAAAATGTTCCTTCTATTTAGCAGACAAGAATATCACCCACGGCTTCCTCTATCCTCCTGCCAGCAATAGAACATCAGATAGCCAATATGATGCTTTAATTACTAGCAATTTGGTACCTATGTATGAAGAATTCAGAAAAATGTGGGACTACTTCCACAGTGTTCTTCTTATAAAACATGCCACAGAAAGAAATGGAGTAAATGTGGTTAGTGGACCAATATTTGATTATAATTATGATGGCCATTTTGATGCTCCAGATGAAATTACCAAACATTTAGCCAACACTGATGTTCCCATCCCAACACACTACTTTGTGGTGCTGACCAGTTGTAAAAACAAGAGCCACACACCGGAAAACTGCCCTGGGTGGCTGGATGTCCTACCCTTTATCATCCCTCACCGACCTACCAACGTGGAGAGCTGTCCTGAAGGTAAACCAGAAGCTCTTTGGGTTGAAGAAAGATTTACAGCTCACATTGCCCGGGTCCGTGATGTAGAACTTCTCACTGGGCTTGACTTCTATCAGGATAAAGTGCAGCCTGTCTCTGAAATTTTGCAACTAAAGACATATTTACCAACATTTGAAACCACTATTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAANucleotide sequence of NPP121-NPP3-Fc (SEQ ID NO: 28)ATGGAAAGGGACGGATGCGCCGGTGGTGGATCTCGCGGAGGCGAAGGTGGAAGGGCCCCTAGGGAAGGACCTGCCGGAAACGGAASGGACAGGGGACGCTCTCACGCCGCTGAAGCTCCAGGCGACCCTCAGGCCGCTGCCTCTCTGCTGGCTCCTATGGACGTCGGAGAAGAACCCCTGGAATAGGCCGCCAGGGCCAGGACTGCCAAGGACCCCAACACCTACAAGATCATCTCCCTCTTCACTTTCGCCGTCGGAGTCAACATCTGCCTGGGATTCACCGCCGAAAAGCAAGGCAGCTGCAGGAAGAAGTGCTTTGATGCATCATTTAGAGGACTGGAGAACTGCCGGTGTGATGTGGCATGTAAAGACCGAGGTGATTGCTGCTGGGATTTTGAAGACACCTGTGTGGAATCAACTCGAATATGGATGTGCAATAAATTTCGTTGTGGAGAGACCAGATTAGAGGCCAGCCTTTGCTCTTGTTCAGATGACTGTTTGCAGAGGAAAGATTGCTGTGCTGACTATAAGAGTGTTTGCCAAGGAGAAACCTCATGGCTGGAAGAAAACTGTGACACAGCCCAGCAGTCTCAGTGCCCAGAAGGGTTTGACCTGCCACCAGTTATCTTGTTTTCTATGGATGGATTTAGAGCTGAATATTTATACACATGGGATACTTTAATGCCAAATATCAATAAACTGAAAACATGTGGAATTCATTCAAAATACATGAGAGCTATGTATCCTACCAAAACCTTCCCAAATCATTACACCATTGTCACGGGCTTGTATCCAGAGTCACATGGCATCATTGACAATAATATGTATGATGTAAATCTCAACAAGAATTTTTCACTTTCTTCAAAGGAACAAAATAATCCAGCCTGGTGGCATGGGCAACCAATGTGGCTGACAGCAATGTATCAAGGTTTAAAAGCCGCTACCTACTTTTGGCCCGGATCAGAAGTGGCTATAAATGGCTCCTTTCCTTCCATATACATGCCTTACAACGGAAGTGTCCCATTTGAAGAGAGGATTTCTACACTGTTAAAATGGCTGGACCTGCCCAAAGCTGAAAGACCCAGGTTTTATACCATGTATTTTGAAGAACCTGATTCCTCTGGACATGCAGGTGGACCAGTCAGTGCCAGAGTAATTAAAGCCTTACAGGTAGTAGATCATGCTTTTGGGATGTTGATGGAAGGCCTGAAGCAGCGGAATTTGCACAACTGTGTCAATATCATCCTTCTGGCTGACCATGGAATGGACCAGACTTATTGTAACAAGATGGAATACATGACTGATTATTTTCCCAGAATAAACTTCTTCTACATGTACGAAGGGCCTGCCCCCCGCATCCGAGCTCATAATATACCTCATGACTTTTTTAGTTTTAATTCTGAGGAAATTGTTAGAAACCTCAGTTGCCGAAAACCTGATCAGCATTTCAAGCCCTATTTGACTCCTGATTTGCCAAAGCGACTGCACTATGCCAAGAACGTCAGAATCGACAAAGTTCATCTCTTTGTGGATCAACAGTGGCTGGCTGTTAGGAGTAAATCAAATACAAATTGTGGAGGAGGCAACCATGGTTATAACAATGAGTTTAGGAGCATGGAGGCTATCTTTCTGGCACATGGACCCAGTTTTAAAGAGAAGACTGAAGTTGAACCATTTGAAAATATTGAAGTCTATAACCTAATGTGTGATCTTCTACGCATTCAACCAGCACCAAACAATGGAACCCATGGTAGTTTAAACCATCTTCTGAAGGTGCCTTTTTATGAGCCATCCCATGCAGAGGAGGTGTCAAAGTTTTCTGTTTGTGGCTTTGCTAATCCATTGCCCACAGAGTCTCTTGACTGTTTCTGCCCTCACCTACAAAATAGTACTCAGCTGGAACAAGTGAATCAGATGCTAAATCTCACCCAAGAAGAAATAACAGCAACAGTGAAAGTAAATTTGCCATTTGGGAGGCCTAGGGTACTGCAGAAGAACGTGGACCACTGTCTCCTTTACCACAGGGAATATGTCAGTGGATTTGGAAAAGCTATGAGGATGCCCATGTGGAGTTCATACACAGTCCCCCAGTTGGGAGACACATCGCCTCTGCCTCCCACTGTCCCAGACTGTCTGCGGGCTGATGTCAGGGTTCCTCCTTCTGAGAGCCAAAAATGTTCCTTCTATTTAGCAGACAAGAATATCACCCACGGCTTCCTCTATCCTCCTGCCAGCAATAGAACATCAGATAGCCAATATGATGCTTTAATTACTAGCAATTTGGTACCTATGTATGAAGAATTCAGAAAAATGTGGGACTACTTCCACAGTGTTCTTCTTATAAAACATGCCACAGAAAGAAATGGAGTAAATGTGGTTAGTGGACCAATATTTGATTATAATTATGATGGCCATTTTGATGCTCCAGATGAAATTACCAAACATTTAGCCAACACTGATGTTCCCATCCCAACACACTACTTTGTGGTGCTGACCAGTTGTAAAAACAAGAGCCACACACCGGAAAACTGCCCTGGGTGGCTGGATGTCCTACCCTTTATCATCCCTCACCGACCTACCAACGTGGAGAGCTGTCCTGAAGGTAAACCAGAAGCTCTTTGGGTTGAAGAAAGATTTACAGCTCACATTGCCCGGGTCCGTGATGTAGAACTTCTCACTGGGCTTGACTTCTATCAGGATAAAGTGCAGCCTGTCTCTGAAATTTTGCAACTAAAGACATATTTACCAACATTTGAAACCACTATTGGTGGAGGAGGCTCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCGATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGTCGAGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGAGAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTA Nucleotide sequence of hNPP3-hFc-pcDNA3 (SEQ ID NO: 29)GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTTATGGAAAGGGACGGATGCGCCGGTGGTGGATCTCGCGGAGGCGAAGGTGGAAGGGCCCCTAGGGAAGGACCTGCCGGAAACGGAAGGGACAGGGGACGCTCTCACGCCGCTGAAGCTCCAGGCGACCCTCAGGCCGCTGCCTCTCTGCTGGCTCCTATGGACGTCGGAGAAGAACCCCTGGAAAAGGCCGCCAGGGCCAGGACTGCCAAGGACCCCAACACCTACAAGATCATCTCCCTCTTCACTTTCGCCGTCGGAGTCAACATCTGCCTGGGATTCACCGCCGAAAAGCAAGGCAGCTGCAGGAAGAAGTGCTTTGATGCATCATTTAGAGGACTGGAGAACTGCCGGTGTGATGTGGCATGTAAAGACCGAGGTGATTGCTGCTGGGATTTTGAAGACACCTGTGTGGAATCAACTCGAATATGGATGTGCAATAAATTTCGTTGTGGAGAGACCAGATTAGAGGCCAGCCTTTGCTCTTGTTCAGATGACTGTTTGCAGAGGAAAGATTGCTGTGCTGACTATAAGAGTGTTTGCCAAGGAGAAACCTCATGGCTGGAAGAAAACTGTGACACAGCCCAGCAGTCTCAGTGCCCAGAAGGGTTTGACCTGCCACCAGTTATCTTGTTTTCTATGGATGGATTTAGAGCTGAATATTTATACACATGGGATACTTTAATGCCAAATATCAATAAACTGAAAACATGTGGAATTCATTCAAAATACATGAGAGCTATGTATCCTACCAAAACCTTCCCAAATCATTACACCATTGTCACGGGCTTGTATCCAGAGTCACATGGCATCATTGACAATAATATGTATGATGTAAATCTCAACAAGAATTTTTCACTTTCTTCAAAGGAACAAAATAATCCAGCCTGGTGGCATGGGCAACCAATGTGGCTGACAGCAATGTATCAAGGTTTAAAAGCCGCTACCTACTTTTGGCCCGGATCAGAAGTGGCTATAAATGGCTCCTTTCCTTCCATATACATGCCTTACAACGGAAGTGTCCCATTTGAAGAGAGGATTTCTACACTGTTAAAATGGCTGGACCTGCCCAAAGCTGAAAGACCCAGGTTTTATACCATGTATTTTGAAGAACCTGATTCCTCTGGACATGCAGGTGGACCAGTCAGTGCCAGAGTAATTAAAGCCTTACAGGTAGTAGATCATGCTTTTGGGATGTTGATGGAAGGCCTGAAGCAGCGGAATTTGCACAACTGTGTCAATATCATCCTTCTGGCTGACCATGGAATGGACCAGACTTATTGTAACAAGATGGAATACATGACTGATTATTTTCCCAGAATAAACTTCTTCTACATGTACGAAGGGCCTGCCCCCCGCATCCGAGCTCATAATATACCTCATGACTTTTTTAGTTTTAATTCTGAGGAAATTGTTAGAAACCTCAGTTGCCGAAAACCTGATCAGCATTTCAAGCCCTATTTGACTCCTGATTTGCCAAAGCGACTGCACTATGCCAAGAACGTCAGAATCGACAAAGTTCATCTCTTTGTGGATCAACAGTGGCTGGCTGTTAGGAGTAAATCAAATACAAATTGTGGAGGAGGCAACCATGGTTATAACAATGAGTTTAGGAGCATGGAGGCTATCTTTCTGGCACATGGACCCAGTTTTAAAGAGAAGACTGAAGTTGAACCATTTGAAAATATTGAAGTCTATAACCTAATGTGTGATCTTCTACGCATTCAACCAGCACCAAACAATGGAACCCATGGTAGTTTAAACCATCTTCTGAAGGTGCCTTTTTATGAGCCATCCCATGCAGAGGAGGTGTCAAAGTTTTCTGTTTGTGGCTTTGCTAATCCATTGCCCACAGAGTCTCTTGACTGTTTCTGCCCTCACCTACAAAATAGTACTCAGCTGGAACAAGTGAATCAGATGCTAAATCTCACCCAAGAAGAAATAACAGCAACAGTGAAAGTAAATTTGCCATTTGGGAGGCCTAGGGTACTGCAGAAGAACGTGGACCACTGTCTCCTTTACCACAGGGAATATGTCAGTGGATTTGGAAAAGCTATGAGGATGCCCATGTGGAGTTCATACACAGTCCCCCAGTTGGGAGACACATCGCCTCTGCCTCCCACTGTCCCAGACTGTCTGCGGGCTGATGTCAGGGTTCCTCCTTCTGAGAGCCAAAAATGTTCCTTCTATTTAGCAGACAAGAATATCACCCACGGCTTCCTCTATCCTCCTGCCAGCAATAGAACATCAGATAGCCAATATGATGCTTTAATTACTAGCAATTTGGTACCTATGTATGAAGAATTCAGAAAAATGTGGGACTACTTCCACAGTGTTCTTCTTATAAAACATGCCACAGAAAGAAATGGAGTAAATGTGGTTAGTGGACCAATATTTGATTATAATTATGATGGCCATTTTGATGCTCCAGATGAAATTACCAAACATTTAGCCAACACTGATGTTCCCATCCCAACACACTACTTTGTGGTGCTGACCAGTTGTAAAAACAAGAGCCACACACCGGAAAACTGCCCTGGGTGGCTGGATGTCCTACCCTTTATCATCCCTCACCGACCTACCAACGTGGAGAGCTGTCCTGAAGGTAAACCAGAAGCTCTTTGGGTTGAAGAAAGATTTACAGCTCACATTGCCCGGGTCCGTGATGTAGAACTTCTCACTGGGCTTGACTTCTATCAGGATAAAGTGCAGCCTGTCTCTGAAATTTTGCAACTAAAGACATATTTACCAACATTTGAAACCACTATTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGAAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCTATTCTATAGTGTCACCTAAATGCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTAGAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

Example 1

FIGS. 1A-1C comprise graphs illustrating studies of hNPP3 steady stateATP hydrolysis activity.

As illustrated in FIG. 1A, time courses of AMP product formation afteraddition of 50 nM hNPP3 with (from bottom to top) 0.98, 1.95, 3.9, 7.8,15.6, 31.3, 62.5, 125, 250 or 500 μM ATP were analyzed. The enzymereaction was quenched with equal volume of 3 M formic acid at differenttimes and the reaction product, AMP, was quantified by HPLC analysiswith an AMP standard curve. The smooth line through the data points werebest fits to a non-linear enzyme kinetic model with product inhibitionand substrate depletion.

FIG. 1B illustrates steady state ATPase cycling rate comparison hNPP3substrate concentration dependence of initial steady state enzymecycling rate was compared with that measured for hNPP1. ATPase cyclingreaction of both 50 nM hNPP3 and hNPP1 depleted ATP substrate within 1minute at 0.98, 1.95 and 3.9 μM ATP. The uncertainty at these low ATPconcentrations was significant, and thus these three rates were omittedfrom the data set during fitting. The hNPP3 steady state ATPase reactionreached the maximum (k_(cat)) of 2.59(±0.04) s⁻¹ enzyme⁻¹, from theweighted average of the measured rates at 7.8, 15.6, 31.3, 62.5, 125 μMsubstrate. The turnover rate of hNPP1 was 3.46(±0.44) s⁻¹ enzyme⁻¹. TheK_(M) for ATP substrate was estimated to be <8 μM.

FIG. 1C illustrates substrate concentration dependence of the η value.The decreasing η value with substrate concentration for both enzymesindicates that substrate depletion contributes to the non-linearity inthe enzyme reaction time courses much more than product inhibition atlower initial substrate concentrations. The similarity of hNPP3 andhNPP1 η values was consistent with the two enzymes having similarreaction rates and product inhibition.

Example 2 Animal Models

The following non-limiting animal models can be used to test theefficacy of the presently claimed compositions on human diseaseresulting from low pyrophosphate (Phi):

-   1. enpp1asj/asj model of Generalized Arterial Calcification of    Infancy (GACI); Li, et al., 2013, Disease Models & Mech.    6(5):1227-35.-   2. enpp12asj/2asj model of Generalized Arterial Calcification of    Infancy (GACI); Li, et al., 2014, PloS one 9(12):e113542.-   3. ABCC6−/− mouse model of Pseudoxanthoma Elasticum (PXE); Jiang, et    al., 2007, J. Invest. Derm. 127(6):1392-402.-   4. HYP mouse model of X-linked hypophosphatasia (XLH); Liang, et    al., 2009. Calcif. Tissue Int. 85(3):235-46.-   5. LmnaG609G/+ mouse model of Hutchison-Gilford Progeria Syndrome;    Villa-Bellosta, et al., 2013, Circulation 127(24):2442-51.-   6. Tip toe walking (ttw) mouse model of Ossification of the    Posterior Longitudinal Ligament (OPLL) (Okawa, et al., 1998, Nature    Genetics 19(3):271-3; Nakamura, et al., 1999, Human Genetics    104(6):492-7) and osteoarthritis (Bertrand, et al., 2012, Annals    Rheum. Diseases 71(7):1249-53).-   7. Rat model of chronic kidney disease (CKD) on the adenine diet;    Schibler, et al., 1968, Clin. Sci. 35(2):363-72; O'Neill, et al.,    2011, Kidney Int. 79(5):512-7.-   8. Mouse model of chronic kidney disease (CKD) on the adenine diet;    Jia, et al., 2013, BMC Nephrol. 14:116.-   9. 5/6th nephrectomy rat model of CKD; Morrison, 1962, Lab Invest.    11:321-32; Shimamura & Morrison. 1975, Am. J. Pathol. 79(1):95-106.-   10. ENPP1 knockout mouse model of GACI and osteopenia; Mackenzie, et    al., 2012, PloS one 7(2):e32177.

In certain embodiments, there is no rodent model that recapitulates theadult form of the human disease GACI, also referred to in the literatureas Autosomal Recessive Hypohposphatemic Rickets type 2 (ARHR2)(Levy-Litan, et al., 2010, Am. J. Human Gen. 86(2):273-8.

Experimental details on enzymatic activity, quantification of plasmaPPi, micro-CT scans, quantification of plasma pyrophosphate uptake andmouse models of calcification are described in detail in the patentapplications and/or publications PCT/US2016/33236, WO2014126965(relating to PCT Patent Application No. PCT/US2014/015945), and US20150359858, each of which is herein incorporated in its entirety byreference.

Example 3 Production and Purification of ENPP3 Fusion Proteins

ENPP3 is produced by establishing stable transfections in either CHO orHEK293 mammalian cells. The protein can be produced in either adherentor suspension cells. To establish stable cell lines the nucleic acidsequence encoding NPP3 fusion proteins (FIGS. 3-5 & SEQ ID NO:s 1-29)into an appropriate vector for large scale protein production. There area variety of these vectors available from commercial sources and any ofthose can be used.

For example, FIG. 3 illustrates a plasmid map of ENPP1-2-1-exENPP3-Fccloned into the pcDNA3 plasmid with appropriate endonuclease restrictionsites. The protein subdomains are color coded to illustrate the signalsequence, extracellular domain of ENPP3, and Fc domains of the fusionprotein. The amino acid sequence of the cloned protein is also displayedbelow the plasmid map and also color coded to illustrate the domains ofthe fusion protein. The pcDNA3 plasmid containing the desired proteinconstructs can be stably transfected into expression plasmid usingestablished techniques such as electroporation or lipofectamine, and thecells can be grown under antibiotic selection to enhance for stablytransfected cells.

Clones of single, stably transfected cells are then established andscreened for high expressing clones of the desired fusion protein.Screening of the single cell clones for ENPP3 protein expression can beaccomplished in a high-throughput manner in 96 well plates using thesynthetic enzymatic substrate pNP-TMP as previously described for ENPP1(Saunders, et al., 2008, Mol. Cancer Therap, 7(10):3352-62; Albright, etal., 2015, Nat Commun. 6:10006). Upon identification of high expressingclones through screening, protein production can be accomplished inshaking flasks or bio-reactors previously described for ENPP1 (Albright,et al., 2015, Nat Commun. 6:10006).

Purification of ENPP3 can be accomplished using a combination ofstandard purification techniques known in the art. These techniques arewell known in art and are selected from techniques such as columnchromatograph, ultracentrifugation, filtration, and precipitation.Column chromatographic purification is accomplished using affinitychromatography such as protein-A and protein-G resins, metal affinityresins such as nickel or copper, hydrophobic exchange chromatography,and reverse-phase high-pressure chromatography (HPLC) using C8-C14resins. Ion exchange may also be employed, such as anion and cationexchange chromatography using commercially available resins such asQ-sepharose (anion exchange) and SP-sepharose (cation exchange), bluesepharose resin and blue-sephadex resin, and hydroxyapatite resins. Sizeexclusion chromatography using commercially available S-75 and 5200Superdex resins can also be employed, as known in the art. Buffers usedto solubilize the protein, and provide the selection media for the abovedescribed chromatographic steps, are standard biological buffers knownto practitioners of the art and science of protein chemistry.

Some examples of buffers that are used in preparation include citrate,phosphate, acetate, tris(hydroxymethyl)aminomethane, saline buffers,glycine-HCL buffers, Cacodylate buffers, and sodium barbital buffers,which are well known in art. Using a single techniques, or a series oftechniques in combination, and the appropriate buffer systems adjustedto the appropriate one can purify the fusion proteins described togreater than 99% purity from crude material (see, for example, FIG. 2).This figure compares partially purified ENPP3 and the crude startingmaterial side by side on a Coomasie stained polyacrylamide gel after asingle purification step. As demonstrated in FIG. 2, a protein ofmolecular weight slightly greater than 105 kD corresponding to theappropriate molecular weight of ENPP3 was enriched from the crudestarting material displayed in the right lane after a singlepurification step. This material can then be additionally purified usingadditional techniques and/or chromatographic steps as described above,to reach substantially higher purity such as ˜99% purity. In certainembodiments, the purified protein has enzymatic activity comparable tothe enzymatic activity described and demonstrated in FIGS. 1A-1C.

Example 4 Usage of Plasma PPi as a Biomarker

Certain embodiments of the invention contemplate the usage of plasmapyrophosphate as a biomarker to determine which individuals are at riskfor diseases of ectopic calcification of the soft tissues, calcificationof the medial vascular wall, low bone mineral density, osteopenia,stroke, arthritis, and/or hereditary forms of rickets. Plasma PPi hasnot been clinically used to predict individuals at risk for the abovedisorders, as demonstrated by the lack of a plasma PPi test in catalogsof laboratory tests offered by leading clinical laboratories, such asMayo Medical Laboratory (www dot mayomedicallaboratories dotcom/test-catalog/alphabetical/P) or Yale University, or leadingcommercial reference laboratories such as ARUP (ltd dot aruplab dotcom/Search/Browse/P) or The Quest Diagnostics Nichols Institute (www dotspecialtylabs dot com/about_us/).

In certain embodiments, plasma PPi has clinical utility as a predictiveand diagnostic agent to identify individuals at risk for the abovedisorders of calcification, ossification, stroke, osteopenia, low bonemineral density, and/or arthritis.

The measurement of plasma PPi can be accomplished by several publishedmethods including radio-isotopic (Cheung, et al., 1977, Anal. Biochem.83(1):61-3) and fluorescent (Jansen, et al., 2013, PNAS USA110(50):20206-11; Jansen, et al., 2014, Arterioscler. Thromb. Vasc.Biol. 34(9):1985-9). Correct measurement of plasma PPi requires thatplatelets are removed from the plasma and that the whole blood, whencollected, is not hemolyzed. Platelets can be removed from the bloodeither by high speed centrifugation or by ultrafiltration. Removal ofplatelets is required to prevent platelets from releasing PPi and ATPinto the plasma upon activation and degranulation, which willartificially elevate the plasma PPi levels. Hemolysis of whole bloodalso releases ATP into the plasma and falsely elevate the measurement ofplasma PPi. Plasma that has been collected from non-hemolyzed blood andremoved of platelets can be used to reliable measure PPi concentrations,and can provide clinical utility as predictive diagnostic identifyingpatients at risk for the above mentioned disorders.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. An isolated polypeptide of formula (I), or a pharmaceutical salt orsolvate thereof:EXPORT-PROTEIN-Z-DOMAIN-X-Y   (I), wherein: EXPORT is absent, or asignal export sequence or a biologically active fragment thereof;PROTEIN is the extracellular domain of ENPP3 (SEQ ID NO:1) or abiologically active fragment thereof; DOMAIN is selected from the groupconsisting of a human IgG Fc domain and human albumin domain; X and Zare independently absent or a polypeptide comprising 1-20 amino acids;and, Y is absent or is a sequence selected from the group consisting of:(DSS)_(n) (SEQ ID NO:6), (ESS)_(n) (SEQ ID NO:7), (RQQ)_(n) (SEQ IDNO:8), (KR)_(n) (SEQ ID NO:9), (SEQ ID NO:10), (KR)_(n) (SEQ ID NO:11),DSSSEEKFLRRIGRFG (SEQ ID NO:12), EEEEEEEPRGDT (SEQ ID NO:13),APWHLSSQYSRT (SEQ ID NO:14), STLPIPHEFSRE (SEQ ID NO:15), VTKHLNQISQSY(SEQ ID NO:16), E_(n) (SEQ ID NO:17), and D_(n) (SEQ ID NO:18), whereineach occurrence of n is independently an integer ranging from 1 to 20.2. The polypeptide of claim 1, wherein the nuclease domain of thePROTEIN or mutant thereof is absent.
 3. The polypeptide of claim 1,wherein EXPORT is absent or selected from the group consisting of SEQ IDNOs:2-5.
 4. The polypeptide of claim 1, wherein X and Z areindependently selected from the group consisting of: absent, apolypeptide consisting of 20 amino acids, a polypeptide consisting of 19amino acids, a polypeptide consisting of 18 amino acids, a polypeptideconsisting of 17 amino acids, a polypeptide consisting of 16 aminoacids, a polypeptide consisting of 15 amino acids, a polypeptideconsisting of 14 amino acids, a polypeptide consisting of 13 aminoacids, a polypeptide consisting of 12 amino acids, a polypeptideconsisting of 11 amino acids, a polypeptide consisting of 10 aminoacids, a polypeptide consisting of 9 amino acids, a polypeptideconsisting of 8 amino acids, a polypeptide consisting of 7 amino acids,a polypeptide consisting of 6 amino acids, a polypeptide consisting of 5amino acids, a polypeptide consisting of 4 amino acids, a polypeptideconsisting of 3 amino acids, a polypeptide consisting of 2 amino acids,and a polypeptide consisting of 1 amino acid.
 5. The polypeptide ofclaim 1, wherein DOMAIN is a human IgG Fc domain selected from the groupconsisting of IgG1, IgG2, IgG3 and IgG4.
 6. The polypeptide of claim 5,which is selected from the group consisting of SEQ ID NOs:19, 21 and 22.7. The polypeptide of claim 1, wherein DOMAIN is a human albumin domain.8. The polypeptide of claim 7, which is selected from the groupconsisting of SEQ ID NOs:24, 25 and
 26. 9. An isolated polypeptidecomprising a soluble region of ENPP3 and lacking a transmembrane domainand a signal peptide, or a fusion protein thereof, wherein thepolypeptide reduces cellular calcification when administered to asubject suffering from diseases of calcification and ossification. 10.The polypeptide of claim 9, which comprises the extracellular domain ofENPP3 (SEQ ID NO:1) or a biologically active fragment thereof.
 11. Thepolypeptide of claim 10, which consists essentially of SEQ ID NO:1 or abiologically active fragment thereof.
 12. A method of treating orpreventing a disease or disorder associated with pathologicalcalcification or pathological ossification in a subject in need thereof,the method comprising administering to the subject a therapeuticallyeffective amount of at least one isolated polypeptide of claim
 1. 13.The method of claim 12, wherein the disease or disorder comprises atleast one selected from the group consisting of general arterialcalcification of infancy (GACI), idiopathic infantile arterialcalcification (IIAC), pseudoxanthoma elasticum (PXE), OPLL,hypophosphatemic rickets, osteoarthritis, calcification ofatherosclerotic plaques, pseudoxanthoma elasticum, hereditary andnon-hereditary forms of osteoarthritis, ankylosing spondylitis,hardening of the arteries occurring with aging, and calciphylaxisresulting from end stage renal disease (or mineral bone disorder ofchronic kidney disease).
 14. The method of claim 12, wherein thenuclease domain of the PROTEIN or mutant thereof is absent.
 15. Themethod of claim 12, wherein EXPORT is absent or selected from the groupconsisting of SEQ ID Nos:2-5.
 16. The method of claim 12, wherein X andZ are independently selected from the group consisting of: absent, apolypeptide consisting of 20 amino acids, a polypeptide consisting of 19amino acids, a polypeptide consisting of 18 amino acids, a polypeptideconsisting of 17 amino acids, a polypeptide consisting of 16 aminoacids, a polypeptide consisting of 15 amino acids, a polypeptideconsisting of 14 amino acids, a polypeptide consisting of 13 aminoacids, a polypeptide consisting of 12 amino acids, a polypeptideconsisting of 11 amino acids, a polypeptide consisting of 10 aminoacids, a polypeptide consisting of 9 amino acids, a polypeptideconsisting of 8 amino acids, a polypeptide consisting of 7 amino acids,a polypeptide consisting of 6 amino acids, a polypeptide consisting of 5amino acids, a polypeptide consisting of 4 amino acids, a polypeptideconsisting of 3 amino acids, a polypeptide consisting of 2 amino acids,and a polypeptide consisting of 1 amino acid.
 17. The method of claim12, wherein the at least one polypeptide is administered acutely orchronically to the subject.
 18. The method of claim 12, wherein the atleast one polypeptide is administered locally, regionally orsystemically to the subject.
 19. The method of claim 12, wherein DOMAINis a human IgG Fc domain selected from the group consisting of IgG1,IgG2, IgG3 and IgG4.
 20. The method of claim 19, wherein the at leastone polypeptide is selected from the group consisting of SEQ ID NOs:19,21 and
 22. 21. The method of claim 12, wherein DOMAIN is a human albumindomain.
 22. The method of claim 21, wherein the at least one polypeptideis selected from the group consisting of SEQ ID NOs:24, 25 and
 26. 23.The method of claim 12, wherein the subject is a mammal.
 24. The methodof claim 23, wherein the mammal is human.
 25. A method of reducing orpreventing vascular calcification in a subject with low plasmapyrophosphate (PPi) or high serum phosphate (Pi), the method comprisingadministering to the subject a therapeutically effective amount of anisolated recombinant human soluble ENPP3 fragment or fusion proteinthereof, wherein the administered amount raises the level of plasma PPiin the subject to at least about 800 nM.
 26. The method of claim 25,wherein the administered amount raises the level of plasma PPi in thesubject to at least about 1 μM.
 27. The method of claim 26, wherein theadministered amount raises the level of plasma PPi in the subject to atleast about 1.5 μM.
 28. The method of claim 25, wherein the subject hasat least one disease selected from a group consisting of GACI, IIAC,PXE, OPLL, MWVC, ARHR2, ESRD, CKD-MBD, XLH, age related osteopenia, CUAand hypophosphatemic rickets.
 29. The method of claim 25, wherein thesoluble ENPP3 fragment or fusion protein thereof comprises theextracellular domain of ENPP3 (SEQ ID NO:1) or a biologically activefragment thereof.
 30. The method of claim 25, wherein the soluble ENPP3fragment consists essentially of SEQ ID NO:1 or a biologically activefragment thereof.
 31. The method of claim 25, wherein the soluble ENPP3fragment or fusion protein thereof lacks a transmembrane domain and asignal peptide.
 32. A method of treating of a subject having ENPP1deficiency or ENPP1-associated disease, the method comprisingadministering to the subject a therapeutically effective amount of anisolated recombinant human soluble ENPP3 fragment or fusion proteinthereof.
 33. The method of claim 32, wherein the subject has at leastone disease selected from a group consisting of GACI, IIAC, PXE, OPLL,MWVC, ARHR2, ESRD, CKD-MBD, XLH, age related osteopenia, CUA andhypophosphatemic rickets.
 34. The method of claim 32, wherein thesoluble ENPP3 fragment or fusion protein thereof comprises theextracellular domain of ENPP3 (SEQ ID NO:1) or a biologically activefragment thereof.
 35. The method of claim 32, wherein the soluble ENPP3fragment consists essentially of SEQ ID NO:1 or a biologically activefragment thereof.
 36. The method of claim 32, wherein the soluble ENPP3fragment or fusion protein thereof lacks a transmembrane domain and asignal peptide. 37-38. (canceled)